Compositions and methods for xi chromosome reactivation

ABSTRACT

In some aspects, the disclosure relates to the reactivation of inactive X chromosomes (Xi). In some embodiments, the disclosure provides compositions and methods for the reactivation of inactive X chromosomes. In some embodiments, the compositions and methods described by the disclosure may be useful for the treatment of dominant X-linked diseases.

RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. § 120 of U.S.application Ser. No. 15/566,533, filed Oct. 13, 2017, entitled“COMPOSITIONS AND METHODS FOR XI CHROMOSOME REACTIVATION”, which is aNational Stage Application of PCT/US2016/027840, filed Apr. 15, 2016,entitled “COMPOSITIONS AND METHODS FOR XI CHROMOSOME REACTIVATION”,which claims the benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication Ser. No. 62/148,106, entitled “COMPOSITION AND METHODS FORXI CHROMOSOME REACTIVATION”, filed Apr. 15, 2015, the entire contents ofeach of which are incorporated by reference herein.

FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numberGM033977 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE DISCLOSURE

The invention relates to methods for reactivating mammalian inactive Xchromosomes through genetic and pharmacological means.

BACKGROUND OF THE DISCLOSURE

X chromosome inactivation (XCI), the random transcriptional silencing ofone X chromosome in somatic cells of female mammals, is a mechanism thatensures equal expression of X-linked genes in both sexes. XCI isinitiated by Xist, a 17-kb non-coding RNA whose expression during earlyembryogenesis is both necessary and sufficient for silencing. Xistrepresses transcription in cis by coating only the X chromosome fromwhich it is produced. Once Xist has been upregulated during earlydevelopment or differentiation, it continues to be expressed from theinactive X (Xi) even in fully differentiated somatic cells. Prior to theinitiation of XCI, Tsix, an antisense repressor of Xist, blocks Xistupregulation on the future active X chromosome (Xa).

An understanding of the factors and mechanisms involved in XCI isdirectly relevant to certain human diseases (e.g., dominant X-linkeddiseases). For example, loss-of-function mutations in the X-linkedmethyl-CpG binding protein 2 (MECP2) gene lead to the neurodevelopmentaldisorder Rett syndrome (RTT). Most RTT patients are females who areheterozygous for MECP2 deficiency due to random XCI. Therapeutic optionsfor the treatment of dominant X-linked diseases, such as Rett syndrome,remain limited.

Accordingly, there is a need for new compositions and methods oftreatment for dominant X-linked diseases.

SUMMARY OF THE DISCLOSURE

The instant disclosure relates to methods and compositions for thereactivation of inactive X (Xi) chromosomes. In some aspects, themethods and compositions described herein may be useful for thetreatment of dominant X-linked diseases, such as Rett syndrome. Thedisclosure is based, in part, on the discovery that inhibition of Xchromosome inactivating factors (XCIFs) can mediate reactivation ofinactive X chromosomes, re-expression of Xi-linked genes and/or reduceexpression or activity of the Xist.

Accordingly, in some aspects, the disclosure provides a method ofinducing expression of an X-linked gene in a cell having an inactive Xchromosome, the method comprising delivering to the cell an X chromosomeinactivation factor (XCIF) inhibitor in an amount effective for inducingexpression of the X-linked gene.

In some aspects, the disclosure provides a method of treating a subjecthaving a dominant X-linked disease, the method comprising administeringto the subject an X chromosome inactivation factor (XCIF) inhibitor inan amount effective for inducing expression a target X-linked gene. Insome embodiments, the dominant X-linked disease results from a mutatedallele of the X-linked gene, and wherein the inhibitor is administeredin an amount effective for inducing expression of a wild-type allele ofthe X-linked gene.

In some embodiments, the cell is of a subject having a dominant X-linkeddisease resulting from a mutated allele of the X-linked gene. In someembodiments, the X-linked gene is MECP2. In some embodiments, theX-linked gene is MECP2 and the X-linked disease is Rett Syndrome.

In some embodiments, the dominant X-linked disease is selected from thegroup consisting of: X-linked hypophosphatemia, incontinentia pigmentitype 2, Aicardi syndrome, CDK5L syndrome, focal dermal hypoplasia, CHILDsyndrome, Lujan-Fryns syndrome, orofaciodigital syndrome 1, hereditarynephritis (Alport syndrome), Giuffre-Tsukahara syndrome, Goltz syndrome,Fragile X syndrome, Bazex-Dupre-Christol syndrome, Charcot-Marie-Toothdisease, chondrodysplasia punctate, erythropoietic protoporphyria,scapuloperoneal myopathy, and craniofrontonasal dysplasia.

In some embodiments, the XCIF inhibitor selectively inhibits activity ofan X chromosome inactivation factor selected from the group consistingof: ACVR1, AURKA, DNMT1, FBXO8, LAYN, NF1, PDPK1, PYGO1, RNF165, SGK1/2,SOX5, STC1, ZNF426 and C17orf98. In some embodiments, the X chromosomeinactivation factor is PI3K and the XCIF inhibitor is GNE-317 orLY29400. In some embodiments, the X chromosome inactivation factor isPDPK1 and the XCIF inhibitor is OSU-03012 or BX912. In some embodiments,the X chromosome inactivation factor is AURKA and the XCIF inhibitor isVX680, CD532, or MLN8237. In some embodiments, the X chromosomeinactivation factor is SGK1/2 and the XCIF inhibitor is GSK650394. Insome embodiments, the X chromosome inactivation factor is ACVR1 and theXCIF inhibitor is dorsomorphin, K02288 or LDN193189.

In some embodiments, the XCIF inhibitor selectively inhibits activity ofmammalian target of rapamycin (mTOR). In some embodiments, the XCIFinhibitor is rapamycin, KU-0063794, or everolimus.

In some embodiments, the XCIF inhibitor is an inhibitory oligonucleotidehaving a region of complementarity that is complementary with at least 8nucleotides of an mRNA encoded by an XCIF gene. In some embodiments, theinhibitory oligonucleotide is selected from the group consisting of:antisense oligonucleotide, siRNA, shRNA and miRNA. In some embodiments,the inhibitory oligonucleotide is a modified inhibitory oligonucleotide.In some embodiments, the modified inhibitory oligonucleotide comprises abridged nucleotide (e.g., a locked nucleic acid (LNA)), phosphorothioatebackbone, and/or a 2′-OMe modification.

In some embodiments, the method further comprises determining that cellhas a mutant allele of the X-linked gene. In some embodiments, themethod further comprises determining that delivery of the XCIF inhibitorto the cell results in induced expression of the X-linked gene. In someembodiments, the method further comprises determining that delivery ofthe inhibitor to the cell results in induced expression of a wild-typeallele of the X-linked gene. In some embodiments, the method furthercomprises determining that delivery of the XCIF inhibitor to the cellresults in reactivation of an X chromosome. In some embodiments, themethod further comprises determining that delivery of the XCIF inhibitorto the cell results in decreased expression or activity of XIST. In someembodiments, the cell is in vitro. In some embodiments, the cell is in asubject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show identification of factors involved in mammalian XCI.FIG. 1A shows a schematic summary of the shRNA screen. The Xi isdesignated as such due to deletion of Xist on the Xa. FIG. 1B shows H4SVcells expressing an shRNA against one of the 13 candidates or, as acontrol, a non-silencing (NS) shRNA were FACS sorted and GFP-positivecells isolated. For each KD cell line, the percent GFP-positive cellswas expressed as the fold increase relative to that obtained with the NSshRNA, which was set to 1. FIG. 1C shows two-color RNA FISH monitoringexpression of G6pdx and Lamp2 (left) and Pgk1 and Mecp2 (right) in eachof the 13 XCIF KD BMSL2 cell lines. DAPI staining is shown in blue. Theexperiment was performed at least twice, and representative images areshown (top) and the results quantified (bottom) from one experiment.

FIGS. 2A-2D show XCIFs are involved in initiation of XCI in mouseembryonic stem cells. FIG. 2A shows two-color RNA FISH monitoringexpression of G6pdx and Lamp2 (left) and Pgk1 and Mecp2 (right) in the13 XCIF KD ES cell lines following differentiation. DAPI staining isalso shown. Representative images are shown (top) and the resultsquantified (bottom). FIG. 2B shows percentage of alkalinephosphatase-negative single cells in the 13 XCIF KD ES cell lines before(top, undifferentiated) and after (bottom, differentiated) treatmentwith RA. FIG. 2C shows qRT-PCR analysis monitoring expression of Oct4 inthe 13 XCIF KD ES cell lines following treatment with RA. As a control,expression of Oct4 in undifferentiated ES cells is shown and was setto 1. Error bars indicate SD. FIG. 2D shows qRT-PCR analysis of XCIFs inundifferentiated and differentiated mouse ES cells. Expression indifferentiated ES cells was normalized to that observed inundifferentiated cells, which was set to 1. Error bars indicate SD.

FIGS. 3A-3I show XCIFs function by promoting Xist expression and/orlocalization, and DNMT1 is a transcriptional activator of Xist on theXi. FIG. 3A shows qRT-PCR analysis monitoring Xist expression in the 13XCIF KD ES cell lines following differentiation. Expression indifferentiated ES cells was normalized to that obtained with the NSshRNA, which was set to 1. Error bars indicate SE. FIG. 3B shows RNAFISH monitoring localization of Xist in the 13 XCIF KD ES cell linesfollowing differentiation. Cells were categorized as having either atypical Xist cloud or “other” pattern, which includes either the lack ofa detectable Xist signal or presence of two small Xist signals, as inundifferentiated ES cells. FIG. 3C shows RNA FISH monitoring expressionof Xist (top) and Mecp2 (bottom) in BMSL2 cells treated with an Xistlocked nucleic acid antisense oligonucleotide (LNA ASO) or a control LNAASO. FIG. 3D shows ChIP analysis monitoring binding of DNMT1 and POL2 tothe Xist promoter and exon 2 in BMSL2 cells expressing a NS or Dnmt1shRNA. Error bars indicate SD. FIG. 3E shows nuclear run-on assaymonitoring transcription of Xist, Hprt and Tbp in BMSL2 cells expressinga NS or DNMT1 shRNA. FIG. 3F shows qRT-PCR analysis monitoring Xistlevels in BMSL2 cells expressing a NS or Dnmt1 shRNA following treatmentwith actinomycin D. Actin mRNA was used as a normalization control.Error bars indicate SD. FIG. 3G shows qRT-PCR analysis monitoring Xistexpression in MEFs isolated from female Dnmt1+/+ and Dnmt1−/− embryos.Four different litters were analyzed (n=4 mice total per genotype), andthe results were averaged. Expression was normalized to that observed inDnmt1+/+ MEFs, which was set to 1. Error bars indicate SD.*P<0.001(Student's t-test). FIG. 3H shows qRT-PCR monitoring levels of Xist andTsix in H4SV cells expressing a NS or DNMT1 shRNA. Expression wasnormalized to that obtained with the NS shRNA, which was set to 1. Errorbars indicate SD. FIG. 3I shows qRT-PCR analysis monitoring Hprt andXist expression in BMSL2 cells treated in the absence or presence of5-AZA. Expression was normalized to that observed in the absence of5-AZA, which was set to 1. Error bars indicate SD.

FIGS. 4A-4I show reactivation of the Xi-linked Mecp2 gene by smallmolecule XCIF inhibitors. FIGS. 4A-4B show two-color RNA FISH monitoringexpression of Xist and Mecp2 in differentiated mouse ES cells treatedwith DMSO (control or -), OSU-03012 or LY294002 (FIG. 4A), and in BMSL2cells treated with DMSO or GNE-317 (FIG. 4B). Representative images areshown (top) using the higher concentrations of the inhibitors, and theresults quantified (bottom). Yellow arrowheads indicate co-localizingXist and Mecp2 signals; white arrowheads indicate Mecp2 signals notco-localizing with Xist. FIG. 4C shows two-color RNA FISH monitoringXist and Mecp2 expression in mouse cortical neurons treated with DMSO(control or -), OSU-03012, BX912 or LY294002. Representative images areshown (top) and the results quantified (bottom). Arrowheads indicateMecp2 signals. FIG. 4D shows two-color RNA FISH monitoring expression ofXist and Mecp2 in mouse BMSL2 fibroblasts treated with DMSO (control or-) or GSK650394. Representative images are shown (top) and the resultsquantified (bottom). Arrowheads indicate Mecp2 signals. FIG. 4E showsqRT-PCR monitoring expression of Xist (left) and Mecp2 (right) in BMSL2cells treated with DMSO or increasing concentrations of GSK650394 (2.5,5 or 10 PM). FIG. 4F shows two-color RNA FISH monitoring expression ofXist and Mecp2 in BMSL2 cells treated with DMSO or K02288.Representative images are shown (top) and the results quantified(bottom). Arrowheads indicate Mecp2 signals. FIG. 4G shows qRT-PCRmonitoring expression of Xist (left) and Mecp2 (right) in BMSL2 cellstreated with DMSO, K02288 (0.5 μM) or LDN193189 (0.5 μM). FIG. 4H showsTwo-color RNA FISH monitoring Xist and Mecp2 expression in BMSL2 cellstreated with DMSO (control or -), LY294002 or OSU-03012, and at least 6days following removal of the inhibitor. Representative images are shown(top) and the results quantified (bottom). Arrowheads indicate Mecp2signals. FIG. 4I shows qRT-PCR monitoring Xi-linked wild-type MECP2expression in human RTT fibroblasts treated with DMSO (-), 5-azacytidine(5-AZA), BX912, OSU-03012 or VX680. As a control, Xa-linked wild-typeMECP2 expression was monitored in another clonal fibroblast cell linederived from the same RTT patient (lane 1). The arrowhead indicates thewild-type MECP2 qRT-PCR product. GAPDH was monitored as a loadingcontrol. Bottom, schematic of the MECP2 wild-type (wt) and mutant (mut)alleles.

FIGS. 5A-5B show defective XCI in female Stc1−/− MEFs. FIG. 5A showstwo-color RNA FISH monitoring expression of G6pdx and Lamp2 (top) andPgk1 and Mecp2 (bottom) in female Stc1+/+ and Stc1−/− MEFs, and as acontrol male Stc1−/− MEFs. Representative images are shown (top) and theresults quantified (bottom). FIG. 5B shows qRT-PCR analysis monitoringXist expression in MEFs isolated from female Stc1+/+ and Stc1−/−embryos. Four different litters were analyzed (n=4 mice total pergenotype), and the results were averaged. Expression was normalized tothat of the ribosomal gene RPL4, and Xist expression in Stc1+/+ MEFs wasset to 1. Error bars indicate SD. *P<0.001 (Student's t-test).

FIGS. 6A-6G show defective XCI in female Stc1−/− mice is not accompaniedby increased X-linked gene expression. FIG. 6A shows a schematic of theRNA-Seq analysis pipeline. FIG. 6B shows distribution of log 2transformed ratio of X-linked gene expression in MEFs from femaleStc1−/− (KO) and Stc1+/+ (WT) embryos (n=3 per genotype). FIG. 6C showsa box plot of X-linked gene expression (log 2 transformed FPKM) in MEFsfrom female Stc1−/− and Stc1+/+ embryos (n=3 per genotype). Boxed areasspan the first to the third quartile. Whiskers represent 15^(th) and85^(th) percentiles. FIG. 6D shows qRT-PCR analysis monitoringexpression of Mecp2 and Hprt in MEFs from 2 different litters of femaleStc1−/− and Stc1+/+ embryos (n=2 mice total per genotype). The resultswere normalized to those obtained in Stc1+/+ MEFs, which was set to 1.Error bars indicate SE. FIG. 6E shows an immunoblot showing MECP2 andSTC1 levels in female Stc1+/+ and Stc1−/− MEFs (left) or brain tissuefemale Stc1+/+ and Stc1−/− P1 mice (right) (n=3 per genotype).(α-tubulin (TUBA) was monitored as a loading control. FIG. 6F showsqRT-PCR analysis of Stc1, Xist, Mecp2 and Hprt expression in BMSL2 cellsexpressing a NS or STC1 shRNA. The results were normalized to thoseobtained with the NS shRNA, which was set to 1. Error bars indicate SE.FIG. 6G shows an immunoblot showing MECP2 and STC1 levels in BMSL2 cellsexpressing a NS or Stc1 shRNA.

FIGS. 7A-7D show shRNAs targeting an XCIF reactivate the Xi-linked Hprtgene and decrease mRNA levels of the targeted gene. FIG. 7A shows brightfield images showing growth of the 13 XCIF KD H4SV cell lines followingselection in HAT medium. FIG. 7B shows qRT-PCR analysis monitoringtarget gene expression in the 13 XCIF KD H4SV cell lines expressing theshRNA identified in the primary screen. For each gene, knockdownefficiency was determined relative to the level of target geneexpression in the control cell line expressing a non-silencing (NS)shRNA, which was set to 1. Error bars indicate SD.

FIG. 7C shows bright field images showing growth of the 13 XCIF KD H4SVcell lines, expressing a second, unrelated shRNA to that shown in FIG.7A, following selection in HAT medium. FIG. 7D shows qRT-PCR analysismonitoring target gene expression in the 13 XCIF KD H4SV cell linesexpressing a second, unrelated shRNA to that shown in FIG. 7B. Errorbars indicate SD.

FIGS. 8A-8D show additional RNA FISH images and control experimentsrelated to FIGS. 1A-1C. FIG. 8A shows representative two-color RNA FISHimages showing expression of G6pdx and Lamp2 (top) and Pgk1 and Mecp2(bottom) in each of the 13 XCIF KD BMSL2 cell lines. DAPI staining isalso shown. FIG. 8B shows that in BMSL2 cells the Xi and Xa encode twodistinguishable Pgk1 alleles, Pgk1a and Pgk1b, respectively, whichdiffer by a single nucleotide polymorphism within the mRNA.Allele-specific expression of the Xi- and Xa-linked Pgk1 genes in eachof the 13 XCIF KD BMSL2 cell lines was analyzed using a singlenucleotide primer-extension (SNuPE) assay. The ratio of Pgk1a:Pgk1bexpression was calculated and normalized to that obtained with the NSshRNA, which was set to 1. The results show that in each of the 13 XCIFKD BMSL2 cell lines the ratio of Pgk1a to Pgk1b was increased,indicating that knockdown of each of the 13 XCIFs reactivated theXi-linked Pgk-1a gene. FIG. 8C shows that in BMSL2 cells the Xi and Xaencode two distinguishable Pgk1 alleles, Pgk1a and Pgk1b, respectively,which differ by a single nucleotide polymorphism within the mRNA.Allele-specific expression of the Xi- and Xa-linked Pgk1 genes in sixrepresentative XCIF KD BMSL2 cell lines was analyzed using a singlenucleotide primer extension (SNuPE) assay. The data are plotted as thefunction of ΔRn for each sample, which represents the reporterfluorescence for each allele (VIC/FAM) normalized to the passive dye.The results show that in each of the six XCIF KD BMSL2 cell lines theXi-linked Pgk1a gene was reactivated. FIG. 7D shows X chromosomepainting experiments in the 13 XCIF KD BMSL2 cell lines. The resultsshow that the X chromosome content of the XCIF KD BMSL2 cell lines wassimilar to that of the control BMSL2 cell line expressing a NS shRNA.Thus, the substantially increased bi-allelic expression of X-linkedgenes observed by RNA FISH in the XCIF KD cell lines cannot be explainedby differences in X chromosome number.

FIGS. 9A-9C show additional RNA FISH images and control experimentsrelated to FIGS. 2A-2D. FIG. 9A shoes representative two-color RNA FISHimages monitoring expression of G6pdx and Lamp2 (top) and Pgk1 and Mecp2(bottom) in the 13 XCIF KD ES cell lines following differentiation. DAPIstaining is also shown. FIG. 9B shows X chromosome painting experimentsin the 13 XCIF KD ES cell lines following differentiation. FIG. 9C showsqRT-PCR analysis monitoring expression of Eomes, Tcf7l2 and Cdx2 in the13 XCIF KD ES cell lines following treatment with RA. As a control,expression of each gene in undifferentiated ES cells is shown and wasset to 1. Error bars indicate SD.

FIGS. 10A-10C show RNA FISH images and control experiments related toFIGS. 3A-3I. FIG. 10A shows RNA FISH images. In each of the 13 XCIF KDES cell lines following differentiation, the majority of cells that lostthe typical Xist localization pattern lacked a detectable Xist signal(see FIG. 3B). However, some cells that had lost the typical Xistlocalization pattern contained two small Xist signals, reminiscent ofundifferentiated ES cells. Examples of this latter localization patternare shown here. Nuclear signals are indicated in red and denoted byarrowheads; DAPI staining is also shown. FIG. 10B shows qRT-PCR analysismonitoring expression of Xist (left), Tsix (middle) and Dnmt1 (right) inH4SV cells expressing a NS or one of two Dnmt1 shRNAs (Dnmt1-1 orDnmt1-2). For Xist and Tsix expression, a second, unrelated Dnmt1 shRNAto that used in FIG. 3H. Expression was normalized to that obtained withthe control NS shRNA, which was set to 1. Error bars indicate SD. FIG.10C shows qRT-PCR analysis monitoring expression of Xist (left), Tsix(middle) and Dnmt1 (right) in differentiated ES cells expressing a NSshRNA or one of two Dnmt1 shRNAs (Dnmt1-1 or Dnmt1-2). Expression wasnormalized to that obtained with the control NS shRNA, which was setto 1. Error bars indicate SD.

FIGS. 11A-11C show additional RNA FISH images related to FIGS. 4A-4E.FIG. 11A and FIG. 11B show two-color RNA FISH monitoring expression ofXist and Mecp2 in differentiated ES cells treated with DMSO (control),OSU-03012 (4 μM) or LY294002 (10 μM) (FIG. 11A), and in BMSL2 cellstreated with DMSO or GNE-317 (5 μM) (FIG. 11B). Yellow boxes indicatecells with co-localizing Xist and Mecp2 signals; white boxes indicatecells with biallelic expression of Mecp2 and complete loss of the Xistsignal. FIG. 11C shows two-color RNA FISH monitoring Xist and Mecp2expression in BMSL2 cells treated with DMSO (control), OSU-03012 (2.5μM) or LY294002 (8 μM), and at least 6 days following removal of theinhibitor. White boxes indicate cells with biallelic expression ofMecp2.

FIGS. 12A-12B show control experiment and RNA FISH images related toFIG. 5. FIG. 12A shows X chromosome painting experiments in femaleStc1+/+ and Stc1−/− MEFs. The results show that the X chromosome contentof Stc1−/− MEFs was similar to that of Stc1+/+ MEFs. Thus, thesubstantially increased bi-allelic expression of X-linked genes observedby RNA FISH in the Stc1−/− MEFs cannot be explained by differences in Xchromosome number. FIG. 12B shows defective XCI in cortical neurons frombrain sections of female Stc1−/− mice. Two-color RNA FISH monitoringexpression of Xist and Mecp2 or G6pdx in cortical neurons from adjacent5-μm brain sections of female Stc1−/− and Stc1+/+ mice (n=3 pergenotype, stage P1). Boxed regions denote cells with two Mecp2 or G6pdxsignals; yellow boxes indicate cells with co-localizing Xist andMecp2/G6pdx signals. All cells in the regions shown represent neuronsthat, based on anatomical landmarks, are present in post-hybridizedsections.

FIGS. 13A-13E show additional experiments and data analyses related toFIGS. 6A-6G. FIG. 13A shows a volcano plot showing distribution of log 2transformed ratio of X-linked gene expression in MEFs isolated fromStc1−/− (KO) and Stc1+/+ (WT) embryos (n=3 per genotype). The genes areplotted against negative transformed log of P value. Red circlesrepresent genes with a >2-fold change in expression and P<0.01. Theresults show that the similarity of X-linked gene expression betweenfemale Stc1+/+ and Stc1−/− MEFs was statistically significant. FIG. 13Bshows box plots displaying changes in autosomal gene expression (log 2transformed FPKM) in Stc1−/− and Stc1+/+ MEFs. Boxed areas span thefirst to the third quartile. Whiskers represent 15^(th) and 85^(th)percentiles; samples falling outside these percentiles are shown ascircles. FIG. 13C shows XCIFs are not generally required for repressionof imprinted genes. Primary female mouse embryonic fibroblasts from thestrain C57BL/6 (CAST7), which contains chromosome 7 from Mus castaneus(Cast), were transduced with shRNAs against each of the XCIFs andanalyzed for allele-specific expression of four genes located onchromosome 7 that are either paternally expressed, (Kcnq1ot1 and Peg3)or maternally expressed (Ascl2 and Zim1). Expression of the two allelescan be distinguished by allele-specific restriction enzyme digestionfollowing gene-specific RT-PCR. The sizes of the undigested and digestedbands are indicated, and the sizes of the predicted digested fragmentsare shown in the table (bottom). If knockdown of an XCIF results inreactivation of the normally silenced allele, a mixture of the maternaland paternal allele-specific digestion patterns would be observed. Theresults show that in all 13 XCIF KD cell lines, all four genes displayedonly the expected allele-specific expression pattern, indicating thatthe XCIFs are not generally required for repression of the imprintedgenes. FIG. 13D shows involvement of Polycomb subunits EZH2 and BMI1 forrepression of the X-linked Hprt gene. (Left) qRT-PCR analysis monitoringHprt expression in BMSL2 cells expressing an Ezh2 or Bmi1 shRNA or, as acontrol, a NS shRNA. (Right) qRT-PCR analysis confirming target geneknockdown in mouse ES cells expressing an Ezh2 (left) or Bmi1 (right)shRNA. Error bars indicate SD. FIG. 13E shows analysis of availabledatasets from Yildirim et al. 2013 showing the distribution of log 2transformed ratio of X-linked gene expression in hematopoietic cellsfrom female heterozygous (HET) Xist mutant mice and wild-type (WT) mice.The data were downloaded from Gene Expression Omnibus (GSE43961),normalized by RMA and filtered by detection above background (DABG)(cutoff P-value<0.0001) using Bioconductor package xps. The percentageof X-linked genes upregulated >1.5-fold is shown.

FIG. 14 shows a schematic diagram of downstream targets of3-phosphoinositide dependent protein kinase-1 (PDPK1).

FIG. 15 shows treatment of mouse fibroblasts with an mTOR inhibitorreactivates the Xi-linked Mecp2 gene. Relative expression of Xist andMecp2 in mouse fibroblasts was measured after treatment with rapamycin,KU-0063794, or everolimus (left). Mecp2 RNA was measured by fluorescencein situ hybridization (FISH) and percentage of nuclei stained wasquantified (right).

FIG. 16 shows treatment of mouse fibroblasts with an mTOR inhibitor(rapamycin, KU-0063794, everolimus) reactivates the Xi-linked Hprt gene,as measured by a hypoxanthine-aminopterin-thymidine (HAT) selectionassay.

FIG. 17 shows inhibition of Aurora kinase A (AURKA) reactivatesXi-linked genes. Relative expression of Xist and Mecp2 in mousefibroblasts was measured after treatment with CD532 and MLN8237. Mecp2RNA was measured by fluorescence in situ hybridization (FISH) andpercentage of nuclei stained was quantified (right). Results wereconfirmed using a HAT selection assay.

FIG. 18 shows treatment of mouse fibroblasts with Activin Receptor Type1 (ACVR1) inhibitor reactivates Xi-linked genes. Relative expression ofXist and Mecp2 in mouse fibroblasts was measured after treatment withK02288, dorsomorphin, or LDN193189. Mecp2 RNA was measured byfluorescence in situ hybridization (FISH) and percentage of nucleistained was quantified (right). Results were confirmed using a HATselection assay.

DETAILED DESCRIPTION

Aspects of the disclosure relate to the biological and pharmacologicalinhibition or reversal of X chromosome inactivation. The disclosure isbased, in part, on the discovery that inhibition of X chromosomeinactivating factors (XCIFs) can mediate reactivation of inactive Xchromosomes, re-expression of X-linked genes and/or reduce expression oractivity of Xist.

In some aspects, the disclosure relates to a method of inducingexpression of an X-linked gene in a cell having an inactive Xchromosome, the method comprising delivering to the cell an X chromosomeinactivation factor (XCIF) inhibitor in an amount effective for inducingexpression of the X-linked gene. As used herein, the term “X chromosomeinactivation factor” refers to a gene or gene product (e.g., a protein)that are required for or involved in maintenance or establishment of Xchromosome inactivation. In some embodiments, inhibition of XCIFexpression and/or activity leads to reactivation of an inactivated Xchromosome or one or more genes residing thereon (Xi-linked genes).Thirteen X chromosome inactivation factors (XCIFs) have been identifiedherein (Table 1), and are indicated as being involved in diverseprocesses including cell signaling (ACVR1, AURKA, NF1, LAYN and PDPK1),cell metabolism (STC1), ubiquitin-dependent regulation (FBXO8 andRNF165) and transcription (PYGO1, SOX5 and ZNF426), for example, asdisclosed in Bhatnagar et al., 2014, Proc Natl Acad Sci USA111:12591-12598.

XCIF Inhibitors

The disclosure relates in part to a discovery of inhibitors of XCIFsthat can reactivate expression of the Xi-linked genes. Inhibitors ofXCIFs can be peptides, proteins, antibodies, small molecules, or nucleicacids. In some embodiments, an XCIF inhibitor selectively inhibitsactivity of an X chromosome inactivation factor selected from the groupconsisting of: ACVR1, AURKA, DNMT1, FBXO8, LAYN, NF1, PIK3, PDPK1,PYGO1, RNF165, SOX5, STC1, ZNF426 and C17orf98.

Aspects of the disclosure relate to inhibition of Activin Receptor Type1 (ACVR1), an XCIF that encodes a receptor serine-threonine kinase (alsoknown as ALK2) that mediates signaling by bone morphogenic proteins(BMPs). Gain-of-function mutations in ACVR1 result in the autosomaldominant disease fibrodysplasia ossificans progressiva (FOP) and havebeen found in the childhood malignancy diffuse intrinsic pontine glioma(DIPG). Several small molecule ACVR1 inhibitors are available, includingK02288 and LDN193189. K02288 is a potent and selective inhibitor of BMPtype 1 receptor signaling; strongly inhibiting ACVR1/ALK2, ALK1, andALK6, and weakly inhibiting the other ALKs and ActRIIA. LDN 193189 is aselective BMP signally inhibitor that inhibits the transcriptionalactivity of the BMP type I receptors ACVR1/ALK2 and ALK3; it alsoexhibits 200-fold selectivity for BMP versus TGF-β. Further examples ofACVR1 inhibitors include LDN19318, DMH-1, ML-347, BML-275, dorsomorphin,and LDN-212854.

Aspects of the disclosure relate to inhibition of Aurora Kinase A(AURKA). In some embodiments, AURKA inhibitors are small molecules.Examples of AURKA inhibitors include but are not limited to VX-680,MLN8237, TAS-119, MLN8054, PF-03814735, SNS-314, BI 811283, AMG 900,AZD1152, AS703569, R763, PHA-739358, CD532, and MK-0457. In someembodiments, the X chromosome inactivation factor is AURKA and the XCIFinhibitor is VX680. In some embodiments, the X chromosome inactivationfactor is AURKA and the XCIF inhibitor is CD532 or MLN8237.

Aspects of the disclosure relate to inhibition of DNA(cytosine-5)-methyltransferase 1 (DNMT1). In some embodiments, DNMT1inhibitors are small molecules. Examples of DNMT1 inhibitors include butare not limited to azacitadine, fazarabine, decitabine, sinefungin,psammaplin A, disulfiram, zebularine, and SGI-1027.

Aspects of the disclosure relate to the inhibition of PI3K/Akt signalingto reactivate Xi-linked genes. In some embodiments, PI3K inhibitors aresmall molecules. Examples of PI3K inhibitors include but are not limitedto GNE317, LY294002, Wortmannin, demethoxyviridin, BEZ235, BGT226,BKM120, BYL719, XL765, XL147, GDC-0941, SF1126, GSK1059615, PX-866,CAL-101, BAY80-6946, GDC-0032, IPI-145, VS-5584, ZSTK474, SAR245409, andRP6530. In some embodiments, the XCIF is PI3K and the XCIF inhibitor isGNE-317 or LY29400.

Aspects of the disclosure relate to inhibition of3-phosphoinositide-dependent protein kinase 1 (PDPK1). In someembodiments, PDPK1 inhibitors are small molecules. Examples of PDPK1inhibitors include but are not limited to OSU-03012, BAG-956, BX-795,GSK-2334470, BX-912, and PHT-427. In some embodiments, the XCIF is PDPK1and the XCIF inhibitor is OSU-03012 or BX912.

The serum and glucocorticoid kinase (SGK) family of serine/threoninekinases includes three distinct but highly homologous isoforms (SGK1,SGK2, and SGK3) that share a similar domain structure. All three areactivated by PDPK1 and have been implicated in a wide variety ofcellular processes and small molecule inhibitors with selectivity forSGKs over AKTs have been developed. Examples of SGK1/2 inhibitorsinclude GSK-650394 and EMD638683.

In some embodiments, an XCIF inhibitor targets a downstream substrate ofPDPK1.

Examples of downstream substrates to PDPK1 include but are not limitedto AKT (also known as protein kinase B), ribosomal protein S6 kinasebeta-1 (S6K1), protein kinase C (PKC), ribosomal s6 kinase (e.g.,p70^(rsk) S6 Kinase), rho-associated, coiled-coil-containing proteinkinase 1 (ROCK1), and mammalian target of rapamycin (mTOR). In someembodiments, an XCIF inhibitor targets mTOR. In some embodiments, anmTOR inhibitor is a small molecule. Examples of mTOR inhibitors includebut are not limited to rapamycin, everolimus, sirolimus, temsirolimus,deforolimus, and KU-0063794.

In some embodiments, the term “small molecule” refers to a synthetic ornaturally occurring chemical compound, for instance a peptide oroligonucleotide that may optionally be derivatized, natural product orany other low molecular weight (often less than about 5 kDalton)organic, bioinorganic or inorganic compound, of either natural orsynthetic origin. Such small molecules may be a therapeuticallydeliverable substance or may be further derivatized to facilitatedelivery.

As used herein the term “inhibitor” or “repressor” refers to any agentthat inhibits, suppresses, represses, or decreases a specific activity,such as the activity of an X chromosome inactivation factors.

In some embodiments, an XCIF inhibitor when delivered to a cellreactivates an inactive X chromosome or one or more genes residingthereon. In some embodiments, delivery of an XCIF inhibitor to a cellresults in an increase in the level of expression of an Xi-linked gene(a gene residing on the inactive X-chromosome) of at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or 500% compared with thelevel of expression of the gene in a control cell that has not beendelivered an XCIF inhibitor. In some embodiments, delivery of an XCIFinhibitor to a cell results in an increase in the level of expression ofan Xi-linked gene (a gene residing in the inactive X-chromosome) in arange of 10% to 50%, 10% to 100%, 10% to 200%, 50% to 500% or morecompared with the level of expression of the gene in a control cell thathas not been delivered an XCIF inhibitor.

Inhibitory Oligonucleotides

In some embodiments, the XCIF inhibitor is an inhibitoryoligonucleotide. Inhibitory oligonucleotides may interfere with geneexpression, transcription and/or translation. Generally, inhibitoryoligonucleotides bind to a target polynucleotide via a region ofcomplementarity. For example, binding of inhibitory oligonucleotide to atarget polynucleotide can trigger RNAi pathway-mediated degradation ofthe target polynucleotide (in the case of dsRNA, siRNA, shRNA, etc.), orcan block the translational machinery (e.g., antisenseoligonucleotides). In some embodiments, inhibitory oligonucleotides havea region of complementarity that is complementary with at least 8nucleotides of an mRNA encoded by an XCIF gene. Inhibitoryoligonucleotides can be single-stranded or double-stranded. In someembodiments, inhibitory oligonucleotides are DNA or RNA. In someembodiments, the inhibitory oligonucleotide is selected from the groupconsisting of: antisense oligonucleotide, siRNA, shRNA and miRNA. Insome embodiments, inhibitory oligonucleotides are modified nucleicacids.

The term “nucleotide analog” or “altered nucleotide” or “modifiednucleotide” refers to a non-standard nucleotide, including non-naturallyoccurring ribonucleotides or deoxyribonucleotides. In some embodiments,nucleotide analogs are modified at any position so as to alter certainchemical properties of the nucleotide yet retain the ability of thenucleotide analog to perform its intended function. Examples ofpositions of the nucleotide which may be derivatized include the 5position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine, 5-propyneuridine, 5-propenyl uridine, etc.; the 6 position, e.g.,6-(2-amino)propyl uridine; the 8-position for adenosine and/orguanosines, e.g., 8-bromo guanosine, 8-chloro guanosine,8-fluoroguanosine, etc. Nucleotide analogs also include deazanucleotides, e.g., 7-deaza-adenosine; O- and N-modified (e.g.,alkylated, e.g., N6-methyl adenosine, or as otherwise known in the art)nucleotides; and other heterocyclically modified nucleotide analogs suchas those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000Aug. 10(4):297-310.

Nucleotide analogs may also comprise modifications to the sugar portionof the nucleotides. For example the 2′ OH-group may be replaced by agroup selected from H, OR, R, F, Cl, Br, I, SH, SR, NH₂, NHR, NR₂, COOR,or OR, wherein R is substituted or unsubstituted C.sub.1-C.sub.6 alkyl,alkenyl, alkynyl, aryl, etc. Other possible modifications include thosedescribed in U.S. Pat. Nos. 5,858,988, and 6,291,438. A locked nucleicacid (LNA), often referred to as inaccessible RNA, is a modified RNAnucleotide. The ribose moiety of an LNA nucleotide is modified with anextra bridge connecting the 2′ oxygen and 4′ carbon.

The phosphate group of the nucleotide may also be modified, e.g., bysubstituting one or more of the oxygens of the phosphate group withsulfur (e.g., phosphorothioates), or by making other substitutions whichallow the nucleotide to perform its intended function such as describedin, for example, Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr.10(2):117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000Oct. 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct.11(5): 317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001Apr. 11(2):77-85, and U.S. Pat. No. 5,684,143. Certain of theabove-referenced modifications (e.g., phosphate group modifications)preferably decrease the rate of hydrolysis of, for example,polynucleotides comprising said analogs in vivo or in vitro. In someembodiments, the inhibitory oligonucleotide is a modified inhibitoryoligonucleotide. In some embodiments, the modified inhibitoryoligonucleotide comprises a locked nucleic acid (LNA), phosphorothioatebackbone, and/or a 2′-OMe modification.

Methods of Treatment

The disclosure relates, in some aspects, to methods useful for thetreatment of certain diseases, such as dominant X-linked diseases. Forexample, loss-of-function mutations in the X-linked methyl-CpG bindingprotein 2 (MECP2) gene lead to the neurodevelopmental disorder Rettsyndrome (RTT).

Accordingly, in some aspects, the disclosure provides a method oftreating a subject having a dominant X-linked disease, the methodcomprising administering to the subject an X chromosome inactivationfactor (XCIF) inhibitor in an amount effective for inducing expression atarget X-linked gene.

Dominant X-linked diseases typically result from a mutated allele of theX-linked gene. The disclosure relates, in part, to XCIF inhibitors thatare effective for inducing expression of a wild-type allele of theX-linked gene. Examples of X-linked diseases and their associatedX-linked genes include Rett syndrome (MECP2), X-linked hypophosphatemia(PHEX), incontinentia pigmenti type 2 (IKBKG), Aicardi syndrome (de novomutation of an X-linked gene), CDK5L syndrome (CDKL5), focal dermalhypoplasia (PORCN), CHILD syndrome (NSDHL), Lujan-Fryns syndrome(MED12), orofaciodigital syndrome 1 (OFD1), hereditary nephritis orAlport syndrome (COL4A3, COL4A4, COL4A5), Giuffre-Tsukahara syndrome(Xp22.13-q21.33), Goltz syndrome (PORCN), Fragile X syndrome (FMR1),Bazex-Dupre-Christol syndrome (Xq24-q27), Charcot-Marie-Tooth disease(GJB1), chondrodysplasia punctata (EBP), erythropoietic protoporphyria(ALAS2), scapuloperoneal myopathy (FLH1), and craniofrontonasaldysplasia (EFNB1).

As used herein, a “subject” is interchangeable with a “subject in needthereof”, both of which may refer to a subject having a dominantX-linked disease, or a subject having an increased risk of developingsuch a disorder relative to the population at large. A subject in needthereof may be a subject having an inactive X chromosome. A subject canbe a human, non-human primate, rat, mouse, cat, dog, or other mammal.

In some aspects, the disclosure provides a method of inducing expressionof an X-linked gene in a cell having an inactive X chromosome, themethod comprising delivering to the cell an X chromosome inactivationfactor (XCIF) inhibitor in an amount effective for inducing expressionof the X-linked gene. In some embodiments, the cell is in vitro. In someembodiments, the cell is in a subject.

As used herein, the terms “treatment”, “treating”, and “therapy” referto therapeutic treatment and prophylactic or preventative manipulations.The terms further include ameliorating existing symptoms, preventingadditional symptoms, ameliorating or preventing the underlying causes ofsymptoms, preventing or reversing causes of symptoms, for example,symptoms associated with a dominant X-linked disease. Thus, the termsdenote that a beneficial result has been conferred on a subject with adisorder (e.g., a dominant X-linked disease), or with the potential todevelop such a disorder. Furthermore, the term “treatment” is defined asthe application or administration of an agent (e.g., therapeutic agentor a therapeutic composition) to a subject, or an isolated tissue orcell line from a subject, who may have a disease, a symptom of diseaseor a predisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisease, the symptoms of disease or the predisposition toward disease.

Therapeutic agents or therapeutic compositions may include a compound ina pharmaceutically acceptable form that prevents and/or reduces thesymptoms of a particular disease (e.g., a dominant X-linked disease).For example a therapeutic composition may be a pharmaceuticalcomposition that prevents and/or reduces the symptoms of a dominantX-linked disease. It is contemplated that the therapeutic composition ofthe present invention will be provided in any suitable form. The form ofthe therapeutic composition will depend on a number of factors,including the mode of administration as described herein. Thetherapeutic composition may contain diluents, adjuvants and excipients,among other ingredients as described herein.

Pharmaceutical Compositions

In some aspects, the disclosure relates to pharmaceutical compositionscomprising an XCIF inhibitor. In some embodiments, the compositioncomprises an XCIF inhibitor and a pharmaceutically acceptable carrier.As used herein the term “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active compound, use thereof in thecompositions is contemplated. Supplementary active compounds can also beincorporated into the compositions. Pharmaceutical compositions can beprepared as described below. The active ingredients may be admixed orcompounded with any conventional, pharmaceutically acceptable carrier orexcipient. The compositions may be sterile.

Typically, pharmaceutical compositions are formulated for delivering aneffective amount of an agent (e.g., an XCIF inhibitor). In general, an“effective amount” of an active agent refers to an amount sufficient toelicit the desired biological response (e.g., reactivation of theinactive X chromosome or one or more genes residing thereon. Aneffective amount of an agent may vary depending on such factors as thedesired biological endpoint, the pharmacokinetics of the compound, thedisease being treated (e.g., a dominant X-linked disease), the mode ofadministration, and the patient.

A composition is said to be a “pharmaceutically acceptable carrier” ifits administration can be tolerated by a recipient patient. Sterilephosphate-buffered saline is one example of a pharmaceuticallyacceptable carrier. Other suitable carriers are well-known in the art.See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Ed. (1990).

It will be understood by those skilled in the art that any mode ofadministration, vehicle or carrier conventionally employed and which isinert with respect to the active agent may be utilized for preparing andadministering the pharmaceutical compositions of the present disclosure.Illustrative of such methods, vehicles and carriers are those described,for example, in Remington's Pharmaceutical Sciences, 4th ed. (1970), thedisclosure of which is incorporated herein by reference. Those skilledin the art, having been exposed to the principles of the disclosure,will experience no difficulty in determining suitable and appropriatevehicles, excipients and carriers or in compounding the activeingredients therewith to form the pharmaceutical compositions of thedisclosure.

An effective amount, also referred to as a therapeutically effectiveamount, of a compound (for example, an antisense nucleic acid (e.g.oligonucleotide) or small molecule capable of inhibiting an XCIF) is anamount sufficient to ameliorate at least one adverse effect associatedwith expression, or reduced expression, of the gene in a cell or in anindividual in need of such modulation. The therapeutically effectiveamount to be included in pharmaceutical compositions depends, in eachcase, upon several factors, e.g., the type, size and condition of thepatient to be treated, the intended mode of administration, the capacityof the patient to incorporate the intended dosage form, etc. Generally,an amount of active agent is included in each dosage form to providefrom about 0.1 to about 250 mg/kg, and preferably from about 0.1 toabout 100 mg/kg. One of ordinary skill in the art would be able todetermine empirically an appropriate therapeutically effective amount.

Combined with the teachings provided herein, by choosing among thevarious active compounds and weighing factors such as potency, relativebioavailability, patient body weight, severity of adverse side-effectsand selected mode of administration, an effective prophylactic ortherapeutic treatment regimen can be planned which does not causesubstantial toxicity and yet is entirely effective to treat theparticular subject. The effective amount for any particular applicationcan vary depending on such factors as the disease or condition beingtreated, the particular therapeutic agent being administered, the sizeof the subject, or the severity of the disease or condition. One ofordinary skill in the art can empirically determine the effective amountof a particular nucleic acid and/or other therapeutic agent withoutnecessitating undue experimentation.

In some cases, compounds of the disclosure are prepared in a colloidaldispersion system. Colloidal dispersion systems include lipid-basedsystems including oil-in-water emulsions, micelles, mixed micelles, andliposomes. In some embodiments, a colloidal system of the disclosure isa liposome. Liposomes are artificial membrane vessels which are usefulas a delivery vector in vivo or in vitro. It has been shown that largeunilamellar vesicles (LUVs), which range in size from 0.2-4.0 m canencapsulate large macromolecules. RNA, DNA and intact virions can beencapsulated within the aqueous interior and be delivered to cells in abiologically active form. Fraley et al. (1981) Trends Biochem Sci 6:77.

Liposomes may be targeted to a particular tissue by coupling theliposome to a specific ligand such as a monoclonal antibody, sugar,glycolipid, or protein. Ligands which may be useful for targeting aliposome to, for example, an smooth muscle cell include, but are notlimited to: intact or fragments of molecules which interact with smoothmuscle cell specific receptors and molecules, such as antibodies, whichinteract with the cell surface markers of cancer cells. Such ligands mayeasily be identified by binding assays well known to those of skill inthe art. In still other embodiments, the liposome may be targeted to atissue by coupling it to an antibody known in the art.

Lipid formulations for transfection are commercially available fromQIAGEN, for example, as EFFECTENE™ (a non-liposomal lipid with a specialDNA condensing enhancer) and SUPERFECT™ (a novel acting dendrimerictechnology).

Liposomes are commercially available from Gibco BRL, for example, asLIPOFECTIN™ and LIPOFECTACE™, which are formed of cationic lipids suchas N-[1-(2, 3 dioleyloxy)-propyl]-N, N, N-trimethylammonium chloride(DOTMA) and dimethyl dioctadecylammonium bromide (DDAB). Methods formaking liposomes are well known in the art and have been described inmany publications. Liposomes also have been reviewed by Gregoriadis G(1985) Trends Biotechnol 3:235-241.

Certain cationic lipids, including in particular N-[1-(2, 3dioleoyloxy)-propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP), maybe advantageous when combined with the XCIF inhibitors of thedisclosure.

In some aspects of the disclosure, the use of compaction agents may alsobe desirable. Compaction agents also can be used alone, or incombination with, a biological or chemical/physical vector. A“compaction agent”, as used herein, refers to an agent, such as ahistone, that neutralizes the negative charges on the nucleic acid andthereby permits compaction of the nucleic acid into a fine granule.Compaction of the nucleic acid facilitates the uptake of the nucleicacid by the target cell. The compaction agents can be used alone, e.g.,to deliver an XCIF inhibitor in a form that is more efficiently taken upby the cell or, in combination with one or more of the above-describedcarriers.

Other exemplary compositions that can be used to facilitate uptake of anXCIF inhibitor include calcium phosphate and other chemical mediators ofintracellular transport, microinjection compositions, electroporationand homologous recombination compositions (e.g., for integrating anucleic acid into a preselected location within the target cellchromosome).

The compounds may be administered alone (e.g., in saline or buffer) orusing any delivery vehicle known in the art. For instance the followingdelivery vehicles have been described: cochleates; Emulsomes; ISCOMs;liposomes; live bacterial vectors (e.g., Salmonella, Escherichia coli,Bacillus Calmette-Gudrin, Shigella, Lactobacillus); live viral vectors(e.g., Vaccinia, adenovirus, Herpes Simplex); microspheres; nucleic acidvaccines; polymers (e.g., carboxymethylcellulose, chitosan); polymerrings; proteosomes; sodium fluoride; transgenic plants; virosomes; and,virus-like particles.

The formulations of the disclosure are administered in pharmaceuticallyacceptable solutions, which may routinely contain pharmaceuticallyacceptable concentrations of salt, buffering agents, preservatives,compatible carriers, adjuvants, and optionally other therapeuticingredients.

The term pharmaceutically-acceptable carrier means one or morecompatible solid or liquid filler, diluents or encapsulating substanceswhich are suitable for administration to a human or other vertebrateanimal. The term carrier denotes an organic or inorganic ingredient,natural or synthetic, with which the active ingredient is combined tofacilitate the application. The components of the pharmaceuticalcompositions also are capable of being commingled with the compounds ofthe present disclosure, and with each other, in a manner such that thereis no interaction which would substantially impair the desiredpharmaceutical efficiency.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

In addition to the formulations described herein, the compounds may alsobe formulated as a depot preparation. Such long-acting formulations maybe formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Suitable liquid or solid pharmaceutical preparation forms are, forexample, aqueous or saline solutions for inhalation, microencapsulated,encochleated, coated onto microscopic gold particles, contained inliposomes, nebulized, aerosols, pellets for implantation into the skin,or dried onto a sharp object to be scratched into the skin. Thepharmaceutical compositions also include granules, powders, tablets,coated tablets, (micro)capsules, suppositories, syrups, emulsions,suspensions, creams, drops or preparations with protracted release ofactive compounds, in whose preparation excipients and additives and/orauxiliaries such as disintegrants, binders, coating agents, swellingagents, lubricants, flavorings, sweeteners or solubilizers arecustomarily used as described above. The pharmaceutical compositions aresuitable for use in a variety of drug delivery systems. For a briefreview of methods for drug delivery, see Langer R (1990) Science249:1527-1533, which is incorporated herein by reference.

The compounds may be administered per se (neat) or in the form of apharmaceutically acceptable salt. When used in medicine the salts shouldbe pharmaceutically acceptable, but non-pharmaceutically acceptablesalts may conveniently be used to prepare pharmaceutically acceptablesalts thereof. Such salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulphuric,nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic,tartaric, citric, methane sulphonic, formic, malonic, succinic,naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts of the carboxylic acid group.

Suitable buffering agents include: acetic acid and a salt (1-2% w/v);citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v);and phosphoric acid and a salt (0.8-2% w/v). Suitable preservativesinclude benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9%w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).

The compositions may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the compounds into associationwith a carrier which constitutes one or more accessory ingredients. Ingeneral, the compositions are prepared by uniformly and intimatelybringing the compounds into association with a liquid carrier, a finelydivided solid carrier, or both, and then, if necessary, shaping theproduct. Liquid dose units are vials or ampoules. Solid dose units aretablets, capsules and suppositories.

Modes of Administration

The pharmaceutical compositions of the present disclosure preferablycontain a pharmaceutically acceptable carrier or excipient suitable forrendering the compound or mixture administrable orally as a tablet,capsule or pill, or parenterally, intravenously, intradermally,intramuscularly or subcutaneously, or transdermally.

The pharmaceutical compositions containing an XCIF inhibitor and/orother compounds can be administered by any suitable route foradministering medications. A variety of administration routes areavailable. The particular mode selected will depend, of course, upon theparticular agent or agents selected, the particular condition beingtreated, and the dosage required for therapeutic efficacy. The methodsof this disclosure, generally speaking, may be practiced using any modeof administration that is medically acceptable, meaning any mode thatproduces therapeutic effect without causing clinically unacceptableadverse effects. Various modes of administration are discussed herein.For use in therapy, an effective amount of the XCIF inhibitor and/orother therapeutic agent can be administered to a subject by any modethat delivers the agent to the desired surface, e.g., mucosal, systemic.

Administering the pharmaceutical composition of the present disclosuremay be accomplished by any means known to the skilled artisan. Routes ofadministration include but are not limited to oral, parenteral,intravenous, intramuscular, intraperitoneal, intranasal, sublingual,intratracheal, inhalation, subcutaneous, ocular, vaginal, and rectal.Systemic routes include oral and parenteral. Several types of devicesare regularly used for administration by inhalation. These types ofdevices include metered dose inhalers (MDI), breath-actuated MDI, drypowder inhaler (DPI), spacer/holding chambers in combination with MDI,and nebulizers.

For oral administration, the compounds can be formulated readily bycombining the active compound(s) with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the compounds ofthe disclosure to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a subject to be treated. Pharmaceutical preparations fororal use can be obtained as solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Optionally the oralformulations may also be formulated in saline or buffers forneutralizing internal acid conditions or may be administered without anycarriers.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. Microspheres formulatedfor oral administration may also be used. Such microspheres have beenwell defined in the art. All formulations for oral administration shouldbe in dosages suitable for such administration. For buccaladministration, the compositions may take the form of tablets orlozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent disclosure may be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds, when it is desirable to deliver them systemically, may beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active compounds may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal or vaginal compositionssuch as suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the compounds, increasing convenience to the subjectand the physician. Many types of release delivery systems are availableand known to those of ordinary skill in the art. They include polymerbase systems such as poly(lactide-glycolide), copolyoxalates,polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyricacid, and polyanhydrides. Microcapsules of the foregoing polymerscontaining drugs are described in, for example, U.S. Pat. No. 5,075,109.Delivery systems also include non-polymer systems that are: lipidsincluding sterols such as cholesterol, cholesterol esters and fattyacids or neutral fats such as mono-, di-, and tri-glycerides; hydrogelrelease systems; silastic systems; peptide-based systems; wax coatings;compressed tablets using conventional binders and excipients; partiallyfused implants; and the like. Specific examples include, but are notlimited to: (a) erosional systems in which an agent of the disclosure iscontained in a form within a matrix such as those described in U.S. Pat.Nos. 4,452,775, 4,675,189, and 5,736,152, and (b) diffusional systems inwhich an active component permeates at a controlled rate from a polymersuch as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686.In addition, pump-based hardware delivery systems can be used, some ofwhich are adapted for implantation.

In some embodiments, an inhibitory oligonucleotide can be delivered tothe cells via an expression vector engineered to express the inhibitoroligonucleotide. An expression vector is one into which a desiredsequence may be inserted, e.g., by restriction and ligation, such thatit is operably joined to regulatory sequences and may be expressed as anRNA transcript. An expression vector typically contains an insert thatis a coding sequence for a protein or for a inhibitory oligonucleotidesuch as an shRNA, a miRNA, or an miRNA. Vectors may further contain oneor more marker sequences suitable for use in the identification of cellsthat have or have not been transformed or transfected with the vector.Markers include, for example, genes encoding proteins that increase ordecrease either resistance or sensitivity to antibiotics or othercompounds, genes that encode enzymes whose activities are detectable bystandard assays or fluorescent proteins, etc.

As used herein, a coding sequence (e.g., protein coding sequence, miRNAsequence, shRNA sequence) and regulatory sequences are said to be“operably” joined when they are covalently linked in such a way as toplace the expression or transcription of the coding sequence under theinfluence or control of the regulatory sequences. If it is desired thatthe coding sequences be translated into a functional protein, two DNAsequences are said to be operably joined if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region would be operably joined to a coding sequence ifthe promoter region were capable of effecting transcription of that DNAsequence such that the resulting transcript might be translated into thedesired protein or polypeptide. It will be appreciated that a codingsequence may encode an miRNA, shRNA or miRNA.

The precise nature of the regulatory sequences needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5′ non-transcribed and 5′ non-translatedsequences involved with the initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CAAT sequence, andthe like. Such 5′ non-transcribed regulatory sequences will include apromoter region that includes a promoter sequence for transcriptionalcontrol of the operably joined gene. Regulatory sequences may alsoinclude enhancer sequences or upstream activator sequences as desired.The vectors of the disclosure may optionally include 5′ leader or signalsequences.

In some embodiments, a virus vector for delivering a nucleic acidmolecule is selected from the group consisting of adenoviruses,adeno-associated viruses, poxviruses including vaccinia viruses andattenuated poxviruses, Semliki Forest virus, Venezuelan equineencephalitis virus, retroviruses, Sindbis virus, and Ty virus-likeparticle. Examples of viruses and virus-like particles which have beenused to deliver exogenous nucleic acids include: replication-defectiveadenoviruses, a modified retrovirus, a nonreplicating retrovirus, areplication defective Semliki Forest virus, canarypox virus and highlyattenuated vaccinia virus derivative, non-replicative vaccinia virus,replicative vaccinia virus, Venezuelan equine encephalitis virus,Sindbis virus, lentiviral vectors and Ty virus-like particle. Anothervirus useful for certain applications is the adeno-associated virus. Theadeno-associated virus is capable of infecting a wide range of celltypes and species and can be engineered to be replication-deficient. Itfurther has advantages, such as heat and lipid solvent stability, hightransduction frequencies in cells of diverse lineages, includinghematopoietic cells, and lack of superinfection inhibition thus allowingmultiple series of transductions. The adeno-associated virus canintegrate into human cellular DNA in a site-specific manner, therebyminimizing the possibility of insertional mutagenesis and variability ofinserted gene expression. In addition, wild-type adeno-associated virusinfections have been followed in tissue culture for greater than 100passages in the absence of selective pressure, implying that theadeno-associated virus genomic integration is a relatively stable event.The adeno-associated virus can also function in an extrachromosomalfashion.

In general, other useful viral vectors are based on non-cytopathiceukaryotic viruses in which non-essential genes have been replaced withthe gene of interest. Non-cytopathic viruses include certainretroviruses, the life cycle of which involves reverse transcription ofgenomic viral RNA into DNA with subsequent proviral integration intohost cellular DNA. In general, the retroviruses arereplication-deficient (e.g., capable of directing synthesis of thedesired transcripts, but incapable of manufacturing an infectiousparticle). Such genetically altered retroviral expression vectors havegeneral utility for the high-efficiency transduction of genes in vivo.Standard protocols for producing replication-deficient retroviruses(including the steps of incorporation of exogenous genetic material intoa plasmid, transfection of a packaging cell lined with plasmid,production of recombinant retroviruses by the packaging cell line,collection of viral particles from tissue culture media, and infectionof the target cells with viral particles) are provided in Kriegler, M.,“Gene Transfer and Expression, A Laboratory Manual,” W.H. Freeman Co.,New York (1990) and Murry, E. J. Ed. “Methods in Molecular Biology,”vol. 7, Humana Press, Inc., Clifton, N.J. (1991).

Various techniques may be employed for introducing nucleic acidmolecules of the disclosure into cells, depending on whether the nucleicacid molecules are introduced in vitro or in vivo in a host. Suchtechniques include transfection of nucleic acid molecule-calciumphosphate precipitates, transfection of nucleic acid moleculesassociated with DEAE, transfection or infection with the foregoingviruses including the nucleic acid molecule of interest,liposome-mediated transfection, and the like. Other examples include:N-TER™ Nanoparticle Transfection System by Sigma-Aldrich, FECTOFLY™transfection reagents for insect cells by Polyplus Transfection,Polyethylenimine “Max” by Polysciences, Inc., Unique, Non-ViralTransfection Tool by Cosmo Bio Co., Ltd., LIPOFECTAMINE™ LTXTransfection Reagent by Invitrogen, SATISFECTION™ Transfection Reagentby Stratagene, LIPOFECTAMINE™ Transfection Reagent by Invitrogen,FUGENE® HD Transfection Reagent by Roche Applied Science, GMP compliantIN VIVO-JETPEI™ transfection reagent by Polyplus Transfection, andInsect GENEJUICE® Transfection Reagent by Novagen.

EXAMPLES

The following examples are intended to illustrate the disclosure. Theyare not meant to limit the disclosure in any way.

Aspects of the present disclosure relate to the reactivation of Xchromosomes. As described herein, small molecule inhibitors of XCIFscan, like RNAi knockdown, reactivate the expression of the Xi-linkedgenes, which has implications for treatment of Rett syndrome and otherdominant X-linked diseases. Thirteen X chromosome inactivation factors(XCIFs) have been identified (Table 1), and are involved in thetranscriptional repression of X-linked genes.

TABLE 1 Summary of X Chromosome Inactivation Factors Chromosome Mousegene Human gene Mouse symbol symbol Gene name (human) Biological processAcvr1 ACVR1 activin A receptor, 2 (2) Signal transduction type 1 AurkaAURKA aurora kinase A 2 (20) Cell cycle regulation Dnmt1 DNMT1 DNA 9(19) Chromatin modification methyltransferase (cytosine-5) 1 Fbxo8 FBXO8F-box protein 8 8 (4) Unknown/Ubiquitin- dependent protein catabolicprocess Layn LAYN Layilin 9 (11) Unknown/Receptor for hyaluronic acidNf1 NF1 neurofibromatosis 1 11 (17) Signal transduction Pdpk1 PDPK13-phosphoinositide 17 (16) Signal transduction dependent proteinkinase-1 Pygo1 PYGO1 pygopus 1 9 (15) Transcriptional regulation Rnf165RNF165 ring finger protein 18 (18) Unknown 165 Sox5 SOX5 SRY-boxcontaining 6 (12) Transcriptional gene 5 regulation Stc1 STC1stanniocalcin 1 14 (8) Cell metabolism Zfp426 ZNF426 zinc finger protein9 (19) Transcriptional 426 regulation 1700001P01Rik C17orf98 RIKEN cDNA11 (17) Unknown 1700001P01 gene

Example 1: Identification of Factors Involved in Mammalian XCI

A previously derived female mouse embryonic fibroblast cell line (H4SV)in which genes encoding green fluorescent protein (GFP) and hypoxanthineguanine phosphoribosyltransferase (HPRT) are present only on the Xi wasused. Knockdown of a factor involved in XCI is expected to reactivateexpression of the Gfp and Hprt reporter genes (FIG. 1A).

A genome-wide mouse shRNA library comprising 62,400 shRNAs was dividedinto 10 pools, which were packaged into retrovirus particles and used totransduce H4SV cells. GFP-positive cells were selected byfluorescence-activated cell sorting (FACS), expanded, and the shRNAswere identified by sequence analysis. To validate the candidates, singleshRNAs directed against each candidate gene were transduced into H4SVcells and the number of GFP-positive cells measured by FACS analysis.The results of these experiments identified 13 candidate genes whoseknockdown resulted in an increased percentage of GFP-positive cellsrelative to that obtained with a control, non-silencing (NS) shRNA (FIG.1B). The cell viability assay of FIG. 7A shows that knockdown of eachcandidate enabled growth in HAT medium, indicating that the Xi-linkedHprt gene was reactivated. As expected, the mRNA levels of the 13candidate genes were decreased in the corresponding KD H4SV cell line(FIG. 7B). To rule out off-target effects, for all 13 candidates it wasshown that a second, unrelated shRNA also reactivated the Xi-linked Hprtgene (FIG. 7C) and decreased mRNA levels of the targeted gene in thecorresponding KD H4SV cell line (FIG. 7D). The 13 X chromosomeinactivation factors (XCIFs) are listed in Table 1 and include proteinsthat are known, or predicted, to be involved in diverse processesincluding cell signaling (PDPK1, AURKA, LAYN, ACVR1 and NF1),transcription (DNMT1, PYGO1, SOX5 and ZFP426) and ubiquitin-dependentregulation (RNF165 and FBXO8). Significantly, DNMT1 has been previouslyshown to be involved in XCI, validating the screening strategy.

To confirm these results, the expression of four X-linked genes, G6pdx,Lamp2, Pgk1 and Mecp2 was analyzed, using two-color RNA fluorescence insitu hybridization (FISH) in BMSL2 cells, an unrelated female mousefibroblast cell line. In BMSL2 cells expressing a control NS shRNA, RNAFISH revealed, as expected, a single nuclear signal for G6pdx, Lamp2,Pgk1 and Mecp2, indicative of monoallelic expression (FIG. 1C and FIG.8A). Knockdown of each of the 13 XCIFs substantially increased thefraction of cells containing two nuclear G6pdx, Lamp2, Pgk1 and Mecp2signals, indicative of biallelic expression. Reactivation of G6pdx,Pgk1, Mecp2 and Hprt in the 13 XCIF KD BMSL2 cell lines was alsodemonstrated by a 1.5-2-fold increase in mRNA levels as monitored byqRT-PCR (FIG. 8B). Reactivation of the Xi-linked Pgk1 gene inrepresentative XCIF KD BMSL2 cell lines was also demonstrated using asingle nucleotide primer extension (SNuPE) assay (FIG. 8C), which coulddistinguish expression of the Xi- and Xa-linked Pgk1 alleles by virtueof a single nucleotide polymorphism. DNA FISH experiments using an Xchromosome-specific paint probe indicated that the X chromosome contentof the XCIF KD BMSL2 cell lines was similar to that of the control BMSL2cell line expressing a NS shRNA (FIG. 8D).

Example 2: The XCIFs are Involved in Initiation of XCI in MouseEmbryonic Stem Cells

Undifferentiated female mouse PGK12.1 ES cells were transduced with aretrovirus expressing an XCIF shRNA. Cells were then treated withretinoic acid (RA), which induces predominantly, but not exclusively,neuronal differentiation. X-linked gene expression was monitored bytwo-color RNA FISH. FIG. 2A and FIG. 9A show that biallelic expressionof the X-linked G6pdx, Lamp2, Pgk1 and Mecp2 genes was substantiallyincreased following knockdown of each XCIF. As above, the X chromosomecontent of the XCIF KD ES cells was similar to that of the control EScell line expressing a NS shRNA (FIG. 9B).

A possible explanation for the failure of one or more of the 13 XCIF KDES cell lines to undergo XCI is that the XCIF is involved indifferentiation. Following RA treatment, differentiation of the 13 XCIFKD ES cell lines was normal, as evidenced by monitoring twowell-established markers of undifferentiated ES cells, alkalinephosphatase activity (FIG. 2B) and Oct4 expression (FIG. 2C). Likewise,several markers of differentiated cells that increase after RA treatment(Eomes [neuronal], Tcf712 [mesoderm] and Cdx2 [epithelial]) wereunaffected by XCIF knockdown (FIG. 9C). Finally, the quantitativereal-time RT-PCR (qRT-PCR) results of FIG. 2D show that expression ofall 13 XCIFs was upregulated following differentiation, explaining, atleast in part, the selective onset of XCI following differentiation.

Example 3: XCIFs Function by Promoting Xist Expression and/orLocalization to the Xi

Following knockdown of the 13 XCIFs in mouse ES cells, RA was added toinduce differentiation and XCI, and Xist expression was analyzed byqRT-PCR. The results of FIG. 3A show that Xist levels were reduced tovarying extents in all XCIF KD ES cell lines. In differentiated femaleES cells, Xist is detected by RNA FISH as a large, diffuse nuclearsignal referred to as a “cloud” that co-localizes with the Xi. FIG. 3Bshows that knockdown of each of the XCIFs reduced to varying extents thepercentage of cells with the Xist localization pattern characteristic ofXCI (see also FIG. 10A). Taken together, these results indicate thatXCIFs promote Xist expression and/or localization of Xist to the Xi.

Several previous studies have suggested that Xist is required for theinitiation but not maintenance of XCI. However, the results of FIGS. 3Aand B implied that Xist was also necessary for maintenance of XCI. Toprovide independent evidence for this model, the Xist function in mouseBMSL2 fibroblasts was abrogated using an Xist antisense locked nucleicacid (LNA) oligonucleotide. The results of FIG. 3C show, consistent withprevious results, that the Xist antisense LNA oligonucleotide perturbedthe normal pattern of Xist expression/localization. Most importantly,the Xist antisense LNA oligonucleotide substantially increased biallelicexpression of X-linked Mecp2. Thus, Xist is involved in both theinitiation and maintenance of XCI.

Example 4: DNMT1 is a Transcriptional Activator of Xist on the Xi

DNMT1, which typically functions as a transcriptional repressor, wasfound to be involved in Xist expression and/or localization to the Xi.To further investigate this finding, chromatin immunoprecipitation(ChIP) experiments were performed in BMSL2 cells in which the Xa harborsa deletion encompassing the Xist promoter and several genes includingHprt. FIG. 3D shows that DNMT1 and, as expected, RNA polymerase II(POL2) were bound near the Xist transcription start-site on the Xi. Thefact that DNMT1 was involved in Xist transcription and bound to the Xistpromoter suggested that DNMT1 might function as a direct transcriptionalactivator of Xist. Consistent with this idea, following knockdown ofDNMT1 the level of POL2 bound to the Xist promoter substantiallydecreased (FIG. 3D). Moreover, in a nuclear run-on assay DNMT1 knockdownreduced Xist transcription but increased Xi-linked Hprt transcription,as expected (FIG. 3E). As a control, transcription of theTATA-box-binding protein (Tbp) gene, which is not X-linked and expressedconstitutively, was unaffected by DNMT1 knockdown. In addition,knockdown of DNMT1 did not affect the half-life of Xist RNA (FIG. 3F)indicating the decreased levels of Xist RNA following DNMT1 depletionwere predominantly transcriptional. Finally, the level of Xisttranscripts was significantly lower in Dnmt1−/− compared to Dnmt1+/+mouse embryonic fibroblasts (MEFs) (FIG. 3G). Collectively, theseresults indicate that DNMT1 is a transcriptional activator of Xist onthe Xi.

The possibility that DNMT1 indirectly activated Xist transcription byrepressing expression of Tsix, which negatively regulates Xist wasconsidered. However, knockdown of DNMT1 in fibroblasts (FIG. 3H and FIG.10B) or murine ES (FIG. 10C) cells substantially decreased Xistexpression but did not affect Tsix levels. DNMT1-mediated methylation atthe Xist promoter could block the binding of a transcriptionalrepressor. Consistent with this possibility, following addition of5-azacytidine, which inhibits DNMT1 enzymatic activity resulting in DNAdemethylation, Xist levels were markedly reduced whereas expression ofthe Xi-linked Hprt gene increased, as expected (FIG. 3I). Collectively,these results suggest that DNMT1 promotes Xist transcription byantagonizing a repressor.

Example 5: Reactivation of the Xi-linked Mecp2 Gene by Small MoleculeXCIF Inhibitors

One of the XCIFs is PDPK1, a serine-threonine kinase that regulatesphosphatidylinositol-3-kinase (PI3K)/AKT signaling. FIG. 4A and FIG. 11Ashow that following treatment of differentiated female mouse ES cellswith a chemical inhibitor of either PDPK1 (OSU-03012) or PI3K(LY294002), there was a dose-dependent loss of the Xist cloud andincreased biallelic expression of Mecp2. Similar results were obtainedin BMSL2 cells using GNE-317 (FIG. 4B and FIG. 11B), a PI3K inhibitorspecifically designed to cross the blood-brain barrier. As expected,with all three inhibitors the majority of cells contained two Mecp2 RNAFISH signals and lacked a detectable Xist cloud. Notably, however, insome cells one of the two Mecp2 RNA FISH signals colocalized with a Xistcloud, which marked the Xi. Similar results were obtained withpost-mitotic mouse cortical neurons using the PDPK1 inhibitors OSU-03012and BX912 or the PI3K inhibitor LY294002 (FIG. 4C).

PDPK1 has a number of known substrates, which are themselves proteinkinases, such as the family of serum- and glucocorticoid-induciblekinases (SGKs). FIG. 4D shows that treatment of BMLS2 cells with theSGK1/2 inhibitor GSK650394 resulted in loss of the Xist cloud andincreased biallelic expression of Mecp2. Consistent with these results,qRT-PCR analysis shows that treatment of BMSL2 cells with GSK650394resulted in a dose-dependent decrease in Xist expression and increase inMecp2 expression (FIG. 4E). Similar results were obtained for twochemical inhibitors of another XCIF, ACVR1: K02288 and LDN193189 (FIGS.4F and 4G).

BMSL2 cells were treated with PDPK1 inhibitor OSU-03012 or PI3Kinhibitor LY294002 resulting in biallelic expression of the Xi-linkedMecp2 gene (FIG. 4H and FIG. 11C). Following removal of the drug for atleast six days, normal Xist expression and localization, and monoallelicexpression of Mecp2, was largely restored, indicating that smallmolecule-mediated reactivation of Xi-linked genes is reversible.

In a clonal fibroblast cell line from an RTT patient, the Xa-linkedmutant MECP2 allele contains a 32 bp deletion, enabling selectivedetection of Xi-linked wild-type MECP2 mRNA in an RT-PCR assay using aprimer within the deleted region. Another clonal fibroblast cell linederived from the same RTT patient in which the wild-type MECP2 allele ison the Xa provided a control for the correct RT-PCR product (FIG. 4I,lane 1). The results show, as expected, that the Xi-linked wild-typeMECP2 allele was not expressed (lane 2) but could be reactivated byaddition of the DNA methyltransferase inhibitor 5-azacytidine (lane 3).Significantly, addition of the PDPK1 inhibitors BX912 and OSU-03012(lanes 4, 5), or VX680 (lane 6), an inhibitor of AURKA, another XCIF(Table 1), reactivated the Xi-linked wild-type MECP2 allele. Thus, XCIFchemical inhibitors can reactivate the Xi-linked Mecp2/MECP2 gene inmurine fibroblasts, ES cells and cortical neurons, as well as human RTTfibroblasts.

Example 6: Defective XCI in Female Stc1−/− Mice

One of the XCIFs isolated in the screen, STC1, is a glycoprotein foundin both the cytoplasm and nucleus. Stc1−/− mice have no obviousphenotype and litters have the expected Mendelian and male:femaleratios. To determine whether STC1 is involved in XCI in the mouse,Stc1+/− mice were intercrossed and the MEFs from the resultant progenywere analyzed by two-color RNA FISH for expression of G6pdx, Lamp2, Pgk1and Mecp2. As expected, female Stc1+/+ MEFs, and as a control maleStc1−/− MEFs, displayed monoallelic expression of G6pdx, Lamp2, Pgk1 andMecp2 (FIG. 5A). By contrast, female Stc1−/− MEFs predominantlydisplayed biallelic expression of the four genes, indicative of an XCIdefect. qRT-PCR analysis revealed reduced Xist levels in female Stc1−/−MEFs compared to female Stc1+/+ MEFs (FIG. 5B). Notably, the Xchromosome content of female Stc1−/− and Stc1+/+ MEFs was comparable(FIG. 12A).

To further validate these findings, Xist and Mecp2, or Xist and G6pdxwere analyzed in cortical neurons from brain sections of Stc1−/− andStc1+/+ post-natal female mice. In female Stc1−/− mice, biallelicexpression of Mecp2 and G6pdx was clearly evident in some corticalneurons (FIG. 12B). Again, in some cells the colocalization of Mecp2 andXist, or G6pdx and Xist signals were observed, indicative ofreactivation of the Xi-linked Mecp2 and G6pdx genes.

Example 7: Defective XCI in Female Stc1−/− Mice is not Accompanied byIncreased X-Linked Gene Expression

Transcriptome profiling (RNA-Seq) experiments were performed todetermine whether the expression levels of X-encoded genes were elevatedin female Stc1−/− MEFs. In these experiments, RNA was prepared fromthree independent cultures of female Stc1+/+ or Stc1−/− MEFs. RNAsamples were processed and amplified followed by high-throughputsequencing (Illumina Hiseq 2000) (FIG. 6A). Sequences were aligned tothe reference genome and bioinformatic analysis of relative X-linkedgene expression was performed. The results of FIG. 6B shows that totalexpression levels of the vast majority (98%) of X-linked genes wereindistinguishable in Stc1+/+ and Stc1−/− MEFs. The similarity ofX-linked gene expression between Stc1+/+ and Stc1−/− MEFs wasstatistically significant (FIG. 6C and FIG. 13A). Moreover, the vastmajority (99%) of autosomal genes were also expressed at statisticallycomparable levels in female Stc1+/+ and Stc1−/− MEFs (FIG. 13B).

To support these RNA-seq-based results, the levels of X-linked genesMecp2 and Hprt were analyzed by qRT-PCR. FIG. 6D shows that Mecp2 andHprt mRNA levels were equivalent in female Stc1+/+ and Stc1−/− MEFs,despite deficient XCI. Furthermore, the immunoblot results of FIG. 6Eshow that the level of MECP2 protein in Stc1+/+ female MEFs (left) andbrain lysates (right) was comparable to that in Stc1−/− females.

The experiments described above were performed in Stc1−/− mice in whichthere was a long-term, stable impairment of XCI. Long-term conditionaldepletion of Xist in mouse hematopoietic cells was shown to not beaccompanied by a general increase in the expression of X-linked genes.To determine whether X-linked gene expression was increased immediatelyfollowing abrogation of XCI, the expression of Mecp2 and Hprt wasanalyzed in mouse BMSL2 fibroblasts following shRNA-mediated knockdownof STC1. In STC1 KD BMSL2 cells there was an approximate two-foldincrease in Mecp2 and Hprt expression, which was evident at both themRNA (FIG. 6F and see FIG. 8B) and protein (FIG. 6G) level.Collectively, these results suggest the existence of a mechanism(s) thatcan compensate for a persistent XCI deficiency to regulate X-linked geneexpression.

Example 8: Reactivation of the Xi-Linked Mecp2 Gene by Small MoleculeInhibition of Downstream Targets of PDPK1

One of the XCIFs is PDPK1, a serine-threonine kinase that regulatesphosphatidylinositol-3-kinase (PI3K)/AKT signaling. PDPK1 has a numberof known substrates, which are themselves protein kinases, such asmammalian target of rapamycin (mTOR), Aurora kinase A (AURKA), andActivin receptor type 1 (ACVR1), as shown in FIG. 14. This exampledescribes treatment with inhibitors of downstream substrates of PDPK1results in reactivation of Xi-linked genes (e.g., Mecp2).

mTOR is a serine-threonine protein kinase that is a downstream componentin PI3K signaling pathways. Mouse fibroblasts were treated with threemTOR inhibitors (rapamycin, KU-0063794, or everolimus) and relativeexpression levels of Xist and Mecp2 were measured. Treatment with eachmTOR inhibitor resulted in a decrease in the relative expression of Xistand an increase in relative expression of Mecp2, indicating reactivationof the Xi-linked Mecp2 gene (FIG. 15). The IC50 of rapamycin,KU-0063794, or everolimus, were measured at 0.1 nm, 10 nm, and 2.4 nm,respectively. Expression of Mecp2 was also analyzed by FISH in BMSL2cells. Two Mcep2 signals were observed in cells treated with the mTORinhibitor, indicating biallelic expression of Mcep2. Thus, treatmentwith each of the mTOR inhibitors reactivates Xi-linked Mecp2 (FIG. 15).

To confirm these results, a hypoxanthine-aminopterin-thymidine (HAT)selection assay was performed. The HAT assay is a dual selection assaythat requires activation of the Xi-linked Hprt gene by an inhibitor withsufficiently low cytotoxicity to allow cellular proliferation andsurvival. Cells containing Xi-linked Hprt were treated with either DMSO(negative control), rapamycin, KU-0063794, or everolimus, and cellulargrowth was measured. Treatment with each mTOR inhibitor but not DMSOresulted in cellular growth, indicating that mTOR inhibitors reactivateXi-linked Hprt gene (FIG. 16).

Aurora kinase A (AURKA) is a serine-threonine kinase that is associatedwith regulation of cell division in the G2-M phases and is a downstreamsubstrate of PDPK1. The human Aurora kinase family comprises threemembers, Aurora kinase A (AURKA), B (AURKB), and C (AURKC). Here, thereactivation of Xi-linked genes using AURKA inhibitors (e.g., VX680,CD532, and MLN 8237) is described.

Mouse fibroblasts were treated with CD532 or MLN 8237 (which havegreater selectivity for AURKA than VX680) and relative expression levelsof Xist and Mecp2 were measured. Treatment with each AURKA inhibitorresulted in a decrease in the relative expression of Xist and anincrease in relative expression of Mecp2, indicating reactivation of theXi-linked Mecp2 gene (FIG. 17). The IC50 of CD532 and MLN 8237 were 45nm and 1.2 nm, respectively. Expression of Mecp2 was also analyzed byFISH in BMSL2 cells. Two Mcep2 signals were observed in cells treatedwith the AURKA inhibitors, indicating biallelic expression of Mcep2.Results were confirmed using HAT selection assay. Thus, treatment witheach of the AURKA inhibitors reactivates Xi-linked Mecp2 (FIG. 17).

Activin receptor type 1 (ACVR1, also known as ALK2) is a receptorserine-threonine kinase that mediates signaling by bone morphogenicproteins. ACVR1 is a downstream substrate of PDPK1. Here, reactivationof Xi-linked genes using ACVR1 inhibitors (e.g., K02288, dorsomorphin,and LDN193189) is described.

Mouse fibroblasts were treated with K02288, dorsomorphin, or LDN193189and relative expression levels of Xist and Mecp2 were measured.Treatment with each ACVR1 inhibitor resulted in a decrease in therelative expression of Xist and an increase in relative expression ofMecp2, indicating reactivation of the Xi-linked Mecp2 gene (FIG. 18).The IC50 of K02288, dorsomorphin, and LDN193189 were 1 nm, 200 nm, and 5nm, respectively. Expression of Mecp2 was also analyzed by FISH in BMSL2cells. Two Mcep2 signals were observed in cells treated with the ACVR1inhibitors, indicating biallelic expression of Mcep2. Results wereconfirmed using HAT selection assay. Thus, treatment with each of theACVR1 inhibitors reactivates Xi-linked Mecp2 (FIG. 18).

Example 9: CRISPR/Cas9-Based Screen to Identify New XCIFs

A CRISPR/Cas9-based screen has been conducted to identify new XCIFs.First, BMSL2 cells, female mouse fibroblasts stably expressing Cas9 andselected for blasticidin resistance, were infected with a mouse GeCKO v2CRISPR library (including 100,000 guide RNAs) and then selecting forpuromycin resistance. Next, the clones were subjected to HAT selectionfor one week. Reactivation of X chromosomes is caused by CRISPR-mediatedinactivation of an XCIF. Growth in HAT medium results from expression offunctional HPRT from a reactivated X chromosome. Guide RNAs wereidentified and validated from positive clones.

Example 10: Materials and Methods Cell Culture

H4SV cells, BMSL2 (HOBMSL2) cells and human RTT fibroblasts werecultured as recommended by the supplier. PGK12.1 cells were cultured aspreviously described and differentiated by replating, on gelatinizedplastic dishes, in the presence of 100 nM alpha-retinoic acid (Sigma)and absence of leukemia inhibitory factor for at least one week.

Isolation of MEFs, Brain Tissue and Cortical Neurons

MEFs were isolated from E8.5 (Dnmt1 mice; Jackson Laboratories) or E14.5(Stc1 mice, provided by D. Sheikh-Hamad) embryos, and were PCR genotypedusing gene-specific and SRY primers (Table 2). Stc1+/+ and Stc1−/− P1pup heads were embedded in O.C.T. compound (Tissue-Tek) and frozen inliquid nitrogen. Brain tissue cryo-sections (5 μm thick) were mounted,fixed and hybridized with FISH probes as described. Neurons wereisolated from the cerebral cortexes of E19.5 C57BL/6 embryos andcultured as described.

Large-Scale shRNA Screen and Validation

The mouse shRNA^(mir) library (release 2.16; Open Biosystems/ThermoScientific) was obtained. H4SV cells (1.1×10⁶) were transduced at amultiplicity of infection of 0.2 with the retroviral pools, generated aspreviously described, and selected for resistance to puromycin for 7days. Cells were FACS sorted and GFP-positive cells were selected.Candidate shRNAs were identified as described previously. To validatethe candidates, 3×10⁵ H4SV or BMSL2 cells were transduced with singleshRNAs and puromycin selected for 4 days. For HAT selection, 3×10⁵ cellswere plated in 6-well plates and selected in medium containing 1×HAT(GIBCO) for 1 week, followed by live cell imaging using a Zeiss Axiovert200 microscope.

RNA FISH

RNA FISH experiments were performed (see Table 2 for cDNA templatesources for probes). Cells were visualized on a Leica DM IRE2 confocalmicroscope. For quantification, 100-500 cells total from at least 10different fields were counted and scored; only cells with a detectableRNA FISH signal were included in the analysis, with the exception of theexperiment in FIG. 3A. Images were adjusted consistently for contrastand brightness using AxioVision Software (Zeiss). All RNA-FISHexperiments were performed at least twice, and representative images andquantification are shown from one experiment.

Alkaline Phosphatase Assay

ES cells were treated in the presence or absence of retinoic acid (seeabove) and analyzed using an Alkaline Phosphatase Staining Kit(Stemgent).

Quantitative Real-Time RT-PCR (qRT-PCR)

Total RNA was isolated and reverse transcribed using Superscript IIReverse Transcriptase (Invitrogen). qRT-PCR was performed as describedpreviously using primers listed in Table 2. For the experiments shown inFIGS. 3F and 3H and FIGS. 10B and C, strand specific cDNA synthesis ofXist and/or Tsix RNAs was performed as described previously, andexpression of Xist and Tsix were normalized to that of Gapdh.

Locked Nucleic Acid (LNA) Nucleofection

Cy3-labeled Xist and control (scrambled) LNAs were added to 10⁴ BMSL2cells at a final concentration of 1 μM in OptiMem using Lipofectamine(Invitrogen) every 6-8 hr for 48 hr.

ChIP Assay

ChIP assays were performed as described previously using extractsprepared 7 days post-retroviral transduction and puromycin selection,and antibodies against DNMT1 or POL2 (Abcam). Primer sequences used foramplifying ChIP products are listed in Table 2.

Nuclear Run-on Assay

Assays were performed in the presence of [P³²]UTP, and radioactive RNAwas isolated using TRIzol reagent. Samples were hybridized to a nylonmembrane immobilized with cDNA probes to Xist (prepared from a plasmidcontaining Xist exons 1 and 6; (51)), Hprt (prepared from a plasmidcontaining the Hprt coding region PCR-amplified using forward5′-TCCGCCTCCTCCTCTGCT-3′ (SEQ ID NO: 114) and reverse5′-GGGAATTTATTGATTTGCAT-3′ (SEQ ID NO: 115) (primers) and Tbp (preparedfrom a cloned Tbp cDNA; Open Biosystems). After washing the membranes,filters were exposed to a PhosphorImager screen and the signal wasquantified on a Fujifilm FLA-7000 imaging system using Image Gauge V4.22Software.

Xist RNA Stability Assay

After treatment with DNase (Ambion), strand-specific Xist RNA levels,and as a control Actin, were quantified by qRT-PCR (see Table 2 forprimer sequences).

Chemical Inhibitor Treatment

Differentiated mouse ES or BMSL2 cells were treated with dimethylsulfoxide (DMSO), LY294002 (Cayman Chemicals; 4 or 10 μM), OSU-03012(Selleck Chemicals; 2.5 or 4 μM), GNE-317 (Genentech Inc., 1.25, 2.5 or5 μM), GSK650394 (Tocris Bioscience, 5 μM), K02288 (Cayman Chemical, 0.5μM), or LDN192189 (Cayman Chemical, 0.5 μM) for 3 days prior to RNA FISHanalysis. For XCI reversibility experiments, BMSL2 cells were treatedwith 8 μM LY294002 or 2.5 μM OSU-03012 for 3 days, washed twice withPBS, and then the media was replaced with fresh media every day for atleast 5 days prior to RNA FISH analysis.

Mouse cortical neurons, isolated as described above, were treated withDMSO, 5 μM BX912 (Axon Medchem), 0.4 μM LY294002 or 2.5 μM OSU-03012 for4 days prior to RNA FISH analysis.

RTT fibroblasts were treated with either DMSO, 5-azacytidine(Calbiochem; 10 μM for 3 days), BX912 (10 μM for 3 days), OSU-03012 (10μM for 2 days followed by 5 μM for 1 day) or VX680 (ChemieTek; 10 μM for2 days followed by 3 μM for 1 day). The wild-type MECP2 levels wereanalyzed as using primers listed in Table 2.

RNA Sequencing and Data Analysis

Total RNA was isolated from MEFs from Stc1+/+ and Stc1−/− embryos (n=3for each genotype) using the RNeasy Plus Mini Kit (Qiagen) and treatedwith RNase-free DNase I (Qiagen). mRNA libraries were generated asdescribed in the TruSeq RNA sample preparation guide (Illumina).

Libraries were sequenced as 50-bp paired ends using an Illumina HiSeq2000. Raw reads (ranging from 47-92 million reads per sample) weretrimmed by removing adaptor sequences and demultiplexed with barcodes.Reads with ambiguous nucleotides and Phred quality scores <46 wereremoved before assembly. Paired-end sequencing reads were aligned usingTopHat (v2.0.6) against mouse genome assembly NCBI38/mm10 (downloadedfrom pre-built indexes at bowtie-bio.sourceforge.net/) by defaultparameters, with the exception of expecting an inner distance betweenmate pairs of 75 bp instead of the default value of 50 bp. The readsaligned by TopHat were processed by Cufflinks (v2.0.1) to assembletranscripts and to measure their relative abundances in FPKM units(fragments per kilobase of exon per million fragments mapped). Assembledtranscripts from control and knockout samples were compared with thetranscriptome downloaded from Ensembl.org and tested for differentialexpression using the Cuffcompare and Cuffdiff utilities in the Cufflinkspackage. Cuffdiff was run with classic-FPKM normalization and a falsediscovery rate (FDR) threshold of 0.05. Genes with a >2-fold change inexpression between Stc1+/+ and Stc1−/− samples and P<0.05 (calculatedusing Cufflinks) were considered significant.

The gene expression results measured by Cufflinks were annotated basedon a GTF file downloaded from Ensembl.org using Bioconductor packageChIPpeakAnno (55). All figures were plotted using R/Bioconductor(v2.15.2) software. The RNA-Seq data have been deposited in NCBI's GeneExpression Omnibus (56) and are accessible to reviewers through GEOSeries accession number GSE47395(ncbi.nlm.nih.gov/geo/query/acc.cgi?token=jtslncmggoemsro&acc=GSE47395).

Immunoblotting

Cell extracts were prepared and immunoblots proved using antibodiesagainst HPRT (Abcam), MECP2 (Abcam), STC1 (Santa Cruz Biotechnology) andα-tubulin.

Single Nucleotide Primer Extension (SNuPE) Assay

A SNuPE assay for Pgk1 was carried out using a Tagman SNP genotypingassay (Applied Biosystems) according to the manufacturer'sspecifications. The following primers and reporters were used for theassay: 5′-CCGGCCAAAATTGATGCTTTCC-3′ (SEQ ID NO: 116),5′-CAGTCCCAAAAGCATCATTGACAT-3′ (SEQ ID NO: 117), 5′-CACTGTCCAAACTAGG-3′(SEQ ID NO: 118) and 5′-CACTGTCCACACTAGG-3′ (SEQ ID NO: 119). The dataare plotted as the function of ΔRn for each sample, which represents thereporter fluorescence for each allele (VIC/FAM) normalized to thepassive reference dye.

Imprinted Gene Analysis

Mouse embryonic fibroblasts from strain C57BL6 (CAST 7), provided by M.Bartolomei, were cultured in DMEM supplemented with 10% fetal calf serumand 10% NEAA. Analysis of imprinted genes was performed using mouseembryonic fibroblasts isolated from the C57BL/6 (CAST7) strain, whichcontains chromosome 7 from the Mus castaneus (Cast) strain in a C57BL/6background. Briefly, total RNA was extracted and cDNA synthesis wascarried out as described above. For PCR amplification, the cDNA wasadded to Ready-To-Go PCR Beads (GE Life Sciences) together with 0.3 μMgene-specific primers (Table 2). Expression of the imprinted gene wasanalyzed by allele-specific restriction enzyme digestion (StcI forAscl2, StuI for Kcnq1ot1, MnlI for Peg3, and FauI for Zim1) and digestedPCR products were resolved by polyacrylamide gel electrophoresis.

TABLE 2List of primers used for qRT-PCR and RT-PCR analysis, cDNA synthesis,ChIP assays, and mouse genotyping; oligo ID numbers for shRNAs; andcDNAs used to prepare RNA FISH probes. Primers qRT-PCRForward primer (5′ → 3′) Reverse primer(s) (5′ → 3′) ActinTTGCCGACAGGATGCAGAA GCCGATCCACACGGAGTACTT (SEQ ID NO: 1) (SEQ ID NO: 43)Acvr1 (mouse) GGCCAGCAGTGTTTTTCTTC TTCCCCTGCTCATAAACCTG (SEQ ID NO: 2)(SEQ ID NO: 44) ACVR1 (human) TCAGGAAGTGGCTCTGGTCT CGTTTCCCTGAACCATGACT(SEQ ID NO: 3) (SEQ ID NO: 45) Aurka (mouse) TAGGATACTGCTTGTTACTTCCTCCAACTGGAGCTGTA (SEQ ID NO: 4) (SEQ ID NO: 46) AURKA (human)TGGAATATGCACCACTTGGA ACTGACCACCCAAAATCTGC SEQ ID NO: 5 (SEQ ID NO: 47)Bmi1 AAATCAGGGGGTTGAAAAATCT GCTAACCACCAATCTTCCTTTG (SEQ ID NO: 6)(SEQ ID NO: 48) Cdx2 GCCAAGTGAAAACCAGGACAAAAGAC GCTGCTGTTGCTGCTGCTGCTTC(SEQ ID NO: 7) (SEQ ID NO: 49) Dnmt1 (mouse) GGAAGGCTACCTGGCTAAAGTCAAGACTGAAAGGGTGTCACTGTCCGAC (SEQ ID NO: 8) (SEQ ID NO: 50) DNMT1 (human)GTGGGGGACTGTGTCTCTGT TGAAAGCTGCATGTCCTCAC (SEQ ID NO: 9) (SEQ ID NO: 51)Eomes CCTGGTGGTGTTTTGTTGTG TTTAATAGCACCGGGCACTC (SEQ ID NO: 10)(SEQ ID NO: 52) Ezh2 CTAATTGGTACTTACTACGA ACTCTAAACTCATACACCTGTCTATAACTTT (SEQ ID NO: 11) CAT (SEQ ID NO: 53) Fbxo8 (mouse)GCTGAGCCATTTTCTTCTCG ATGATGGTTTCTGGCCACTC (SEQ ID NO: 12)(SEQ ID NO: 54) FBXO8 (human) CAAGGGTTGTGGAGAGTGGT ATGTCAATGCCTCCTTGGAC(SEQ ID NO: 13) (SEQ ID NO: 55) Gapdh ATGGCCTTCCGTGTTCCTACATAGGGCCTCTCTTGCTCAG (SEQ ID NO: 14) (SEQ ID NO: 56) G6pdxTCAAAGCACACGCCCTCTT TAGCGCACAGCCAGTTTCC (SEQ ID NO: 15) (SEQ ID NO: 57)Hprt AAGCTTGCTGGTGAAAAGGA TTGCGCTCATCTTAGGCTTT (SEQ ID NO: 16)(SEQ ID NO: 58) Layn (mouse) GCAAGGAGAGTGGATGGGTA ACTTGTGATGCTGTGCTTGC(SEQ ID NO: 17) (SEQ ID NO: 59) LAYN (human) CTACAGGCCGTGCTGCTGCTGACTAGCTGGCCTCCATC (SEQ ID NO: 18) (SEQ ID NO: 60) Mecp2CATGGTAGCTGGGATGTTAGG GCAATCAATTCTACTTTAGA (SEQ ID NO: 19)GCG (SEQ ID NO: 61) Nf1 (mouse) GTAGCCACAGGTCCCTTGTCCTGAGAACAAGTACACAGAGAGTGA (SEQ ID NO: 20) (SEQ ID NO: 62) NF1 (human)AATTCTGCCTCTGGGGTTTT GCTGTTTCCTTCAGGAGTCG (SEQ ID NO: 21)(SEQ ID NO: 63) 0ct4 CTCACCCTGGGCGTTCTCT AGGCCTCGAAGCGACAGA(SEQ ID NO: 22) (SEQ ID NO: 64) Pdpk1 (mouse) GGTCCAGTGGATAAGCGAAATTTCTGCACCACTTGTGAGC (SEQ ID NO: 23) (SEQ ID NO: 65) PDPK1 (human)GACTCTTCCGTGCGTTCTTC GAGGAGAAAGGTGACCCACA (SEQ ID NO: 24)(SEQ ID NO: 66) Pgk1 ATGTCGCTTTCCAACAAGCTG GCTCCATTGTCCAAGCAGAAT(SEQ ID NO: 25) (SEQ ID NO: 67) Pygo1 (mouse) TAATGTCAGCGGAACAGGACTTATCTGGGCTTCCGAGTTG (SEQ ID NO: 26) (SEQ ID NO: 68) PYGO1 (human)ATCCTGGCTTTGGAGGCTAT GTGGCCCAAAGTTAAAAGCA (SEQ ID NO: 27)(SEQ ID NO: 69) Rnf165 (mouse) ATGCCTCCAGCTACAGCCTA GCCCAATGCTAACTGAGAGC(SEQ ID NO: 28) (SEQ ID NO: 70) RNF165 AGGGAGAGCTGGAAAAGGAGAGCCCTCCCTGGTTTAGTGT (human) (SEQ ID NO: 29) (SEQ ID NO: 71)Sox5 (mouse) GTGGAAGAGGAGGAGAGTGAGA AAATTCCTCAGAGTGAGGCTTG(SEQ ID NO: 30) (SEQ ID NO: 72) SOX5 (human) AGGGACTCCCGAGAGCTTAGTTGTTCTTGTTGCTGCTTGG (SEQ ID NO: 31) (SEQ ID NO: 73) Stc1 (mouse)AAGTCATACAGCAGCCCAATCA CCAGAAGGCTTCGGACAAGTC (SEQ ID NO: 32)(SEQ ID NO: 74) STC1 (human) TGATCAGTGCTTCTGCAACC TCACAGGTGGAGTTTTCCAG(SEQ ID NO: 33) (SEQ ID NO: 75) Tcf7l2 AAAACAGCTCCTCCGATTCCTAAAGAGCCCTCCATCTTGC (SEQ ID NO: 34) (SEQ ID NO: 76) TsixCAATCTCGCAAGATCCGGTGA TCAAGATGCGTGGATATCTCGG (TSIX2F) (SEQ ID NO: 35)(P422R) (SEQ ID NO: 77) Xist CCCTGCTAGTTTCCCAATGA GGAATTGAGAAAGGGCACAA(non-strand (SEQ ID NO: 36) (SEQ ID NO: 78) specific) Xist (strandGATGCCAACGACACGTCTGA AAGGACTCCAAAGTAACAAT specific) (XIST2281F) TCA (XIST2424R) (SEQ ID NO: 37) (SEQ ID NO: 79) XIST (human)ACGCTGCATGTGTCCTTAGT ATTTGGAGCCTCTTATAGCTG AGTC (SEQ ID NO: 38)TTTG (SEQ ID NO: 80) Zfp426 ATGACCTTTCGCTCATGGAC GGCAAGCTTTGCTTTAGTGC(mouse) (SEQ ID NO: 39) (SEQ ID NO: 81) ZNF426 CTGAGGTGGGTGGATCACTTCTCTGCTTCCTGGGTTCAAG (human) (SEQ ID NO: 40) (SEQ ID NO: 82)1700001P01Rik GCTGATGTCAACTGTTTCC CGCAGAATCTTCCACCCT (mouse)(SEQ ID NO: 41) (SEQ ID NO: 83) Cl0orf98 TCGGGCAAGGACAAAGATACCGATGGCTATGAAGGGAAAA (human) (SEQ ID NO: 42) (SEQ ID NO: 84) RT-PCRForward primer (5′ → 3′)  Reverse primer(s) (5′ → 3′)  Mecp2CCGATCTGTGCAGGAGACCG TGGGGTCCTCGGAGCTCTCGGGCT (1^(st) round)(SEQ ID NO: 85) (SEQ ID NO: 91) Mecp2 GACCCGGGAGACGGTCAGCAAGCTCTCGGGCTCAGGTGGAGGT (2^(nd) round) (SEQ ID NO: 86) (SEQ ID NO: 92)Ascl2 TGAGCATCCCACCCCCCTA CCAAACATCAGCGTCAGTATAG (SEQ ID NO: 87)(SEQ ID NO: 93) Kncq1ot1 ATTGGGAACTTGGGGTGGAGGC GGCACACGGTATGAGAAAAGATTG(SEQ ID NO: 88) (SEQ ID NO: 94) Peg3 ATGCCCACTCCGTCAGCGGCTCATCCTTGTGAACTTTG (SEQ ID NO: 89) (SEQ ID NO: 95) Zim1CTTCAAGCAGAGCACAAAGC GTGGCACACGAAAGGTTTCTC (SEQ ID NO: 90)(SEQ ID NO: 96) cDNA synthesis XistAGAGCATTACAATTCAAGGCTC (XIST2688R) (SEQ ID NO: 97) TsixGATGCCAACGACACGTCTGA (TSIX2R) (SEQ ID NO: 98) GapdhTGTGAGGGAGATGCTCAGTG (GAPDR) (SEQ ID NO: 99) ChIPForward primer (5′ → 3′) Reverse primer(s)(5′ → 3′) Xist TAAAGGTCCAATAAGATGTCAGAA GGAGAGAAACCACGGAAGAA (promoter)(SEQ ID NO: 100) (SEQ ID NO: 102) Xist  GTGCTCCTGCCTCAAGAAGAAGCACTCTTCACTCCTCTAAATCCAG (exon 2) (SEQ ID NO: 101) (SEQ ID NO: 103)Mouse genotyping Forward primer (5′ → 3′) Reverse primer(s) (5′ → 3′)Dnmt1+/+ CTTGGGCCTGGATCTTGGGGATC GGG CCAGTTGTGTGACTTGG (SEQ ID NO: 104)(SEQ ID NO: 109) Dnmt1−/− GGGAACTTCCTGACTAGGGG GGGCCAGTTGTGTGACTTGG(SEQ ID NO: 105) (SEQ ID NO: 110) Stc1+/+ AGCGCACGAGGCGGAACAAAAGAGAGCCGCTGTGAGGCGT (SEQ ID NO: 106) (SEQ ID NO: 111) Stc1−/−AAAAGCCAGAGGTGCAAGAA TATGATCGGAATTCCTCGAC (SEQ ID NO: 107)(SEQ ID NO: 112) SRY TTGTCTAGAGAGCATGGAGGGCC CCACTCCTCTGTGACACTTTAGCATGTCAA (SEQ ID NO: 108) CCTCCGA (SEQ ID NO: 113) shRNAs Gene Oligo IDAcvr1 V2MM_75565 V2MM_76215 Aurka V2MM_188005 V2MM_71909 Bmi1 V2MM_10594V2MM_2034 Dnmt1 V2MM_46797 V2LMM_43170 Ezh2 V2MM_35988 V2MM_30422 Fbxo8V2MM_36526 V3LMM_494067 Layn V2MM_130482 V2MM_214085 Nf1 V2MM_194180V2HS_76027 Pdpk1 V2MM_75859 V2MM_72465 Pygo1 V2MM_110610 V2MM_110609Rnf165 V2MM_172866 TRCN0000135474 Sox5 V2MM_6385 V2HS_94936 Stc1V2MM_22454 V2MM_26886 TRCN0000109921 Zfp426 V2MM_31994 TRCN00000850161700001P01Rik V2MM_100177 V2MM_205788 cDNAs Gene Clone number* G6pdxBAC clone RP23-13D21 Lamp2 BAC clone RP24-173A8 Mecp2fosmid clone WI1-894A5 or WI1-1269o10 Pgk1 BAC RP23-404E5 Xist —*obtained from the BACPAC Resources Center

OTHER EMBODIMENTS

The description of the specific embodiments of the disclosure ispresented for the purposes of illustration. It is not intended to beexhaustive or to limit the scope of the disclosure to the specific formsdescribed herein. Although the disclosure includes reference to severalembodiments, it will be understood by one of ordinary skill in the artthat various modifications can be made without departing from the spiritand the scope of the disclosure.

All patents, patent applications, and publications referenced herein arehereby incorporated by reference. Other embodiments are in the claims.

1. A method of inducing expression of an X-linked gene in a cell having an inactive X chromosome, the method comprising delivering to the cell an X chromosome inactivation factor (XCIF) inhibitor in an amount effective for inducing expression of the X-linked gene, optionally wherein the cell is of a subject having a dominant X-linked disease.
 2. (canceled)
 3. The method of claim 1, wherein the X-linked gene is MECP2.
 4. (canceled)
 5. The method of claim 1, wherein the dominant X-linked disease is selected from the group consisting of Rett Syndrome, X-linked hypophosphatemia, incontinentia pigmenti type 2, Aicardi syndrome, CDK5L syndrome, focal dermal hypoplasia, CHILD syndrome, Lujan-Fryns syndrome, orofaciodigital syndrome 1, hereditary nephritis (Alport syndrome), Giuffre-Tsukahara syndrome, Goltz syndrome, Fragile X syndrome, Bazex-Dupre-Christol syndrome, Charcot-Marie-Tooth disease, chondrodysplasia punctate, erythropoietic protoporphyria, scapuloperoneal myopathy, and craniofrontonasal dysplasia.
 6. The method of claim 1, wherein the XCIF inhibitor selectively inhibits activity of an X chromosome inactivation factor selected from the group consisting of: ACVR1, AURKA, DNMT1, FBXO8, LAYN, NF1, PI3K, PDPK1, PYGO1, RNF165, SGK1/2, SOX5, STC1, ZNF426 and C17orf98.
 7. The method of claim 6, wherein: (i) the X chromosome inactivation factor is PI3K and the XCIF inhibitor is GNE-317 or LY29400, or (ii) the X chromosome inactivation factor is PDPK1 and the XCIF inhibitor is OSU-03012 or BX912; or, (iii) the X chromosome inactivation factor is AURKA and the XCIF inhibitor is VX680, CD532, or MLN8237; or, (iv) the X chromosome inactivation factor is SGK1/2 and the XCIF inhibitor is GSK650394; or, (v) the X chromosome inactivation factor is ACVR1 and the XCIF inhibitor is K02288, dorsomorphin, or LDN193189. 8-11. (canceled)
 12. The method of claim 1, wherein the XCIF inhibitor selectively inhibits activity of mTOR, optionally wherein the inhibitor is rapamycin, KU-0063794, or everolimus.
 13. (canceled)
 14. The method of claim 1, wherein the XCIF inhibitor is an inhibitory oligonucleotide having a region of complementarity that is complementary with at least 8 nucleotides of an mRNA encoded by an XCIF gene, optionally wherein: (i) the inhibitory oligonucleotide is selected from the group consisting of: antisense oligonucleotide, siRNA, shRNA and miRNA; or, (ii) the inhibitory oligonucleotide comprises one or more modified nucleotides, wherein the one or more of the modified nucleotides is an LNA nucleotide; or, (iii) inhibitory oligonucleotide comprises one or more modified internucleoside linkages. 15-18. (canceled)
 19. The method of claim 1 further comprising determining that the cell has a mutant allele of the X-linked gene.
 20. The method of claim 1 further comprising determining that delivery of the XCIF inhibitor to the cell results in (i) induced expression of the X-linked gene, or a wild-type allele of the X-linked gene; or, (ii) determining that an X-chromosome is reactivated; or (iii) determining that there is decreased expression or activity of XIST. 21-23. (canceled)
 24. The method of claim 1, wherein in the cell is in vitro or in a subject.
 25. (canceled)
 26. A method of treating a subject having a dominant X-linked disease, the method comprising: administering to the subject an X chromosome inactivation factor (XCIF) inhibitor in an amount effective for inducing expression a target X-linked gene.
 27. The method of claim 26, wherein the dominant X-linked disease results from a mutated allele of the X-linked gene, and wherein the inhibitor is administered in an amount effective for inducing expression of a wild-type allele of the X-linked gene.
 28. The method of claim 26, wherein the X-linked gene is MECP2.
 29. (canceled)
 30. The method of claim 28, wherein the dominant X-linked disease is selected from the group consisting of: Rett Syndrome, X-linked hypophosphatemia, incontinentia pigmenti type 2, Aicardi syndrome, CDK5L syndrome, focal dermal hypoplasia, CHILD syndrome, Lujan-Fryns syndrome, orofaciodigital syndrome 1, hereditary nephritis (Alport syndrome), Giuffre-Tsukahara syndrome, Goltz syndrome, Fragile X syndrome, Bazex-Dupre-Christol syndrome, Charcot-Marie-Tooth disease, chondrodysplasia punctate, erythropoietic protoporphyria, scapuloperoneal myopathy, and craniofrontonasal dysplasia.
 31. The method of claim 26, wherein the XCIF inhibitor selectively inhibits activity of an X chromosome inactivation factor selected from the group consisting of: ACVR1, AURKA, DNMT1, FBXO8, LAYN, NF1, PI3K, PDPK1, PYGO1, RNF165, SGK1/2, SOX5, STC1, ZNF426 and C17orf98.
 32. The method of claim 31, wherein: (i) the X chromosome inactivation factor is PI3K and the XCIF inhibitor is GNE-317 or LY29400, or (ii) the X chromosome inactivation factor is PDPK1 and the XCIF inhibitor is OSU-03012 or BX912; or, (iii) the X chromosome inactivation factor is AURKA and the XCIF inhibitor is VX680, CD532, or MLN8237; or, (iv) the X chromosome inactivation factor is SGK1/2 and the XCIF inhibitor is GSK650394; or (v) the X chromosome inactivation factor is ACVR1 and the XCIF inhibitor is K02288, dorsomorphin, or LDN193189. 33-36. (canceled)
 37. The method of claim 26, wherein the XCIF inhibitor selectively inhibits activity of mTOR, optionally wherein the inhibitor is rapamycin, KU-0063794, or everolimus.
 38. (canceled)
 39. The method of claim 26, wherein the XCIF inhibitor is an inhibitory oligonucleotide having a region of complementarity that is complementary with at least 8 nucleotides of an mRNA encoded by an XCIF gene, optionally wherein (i) the inhibitory oligonucleotide is selected from the group consisting of: antisense oligonucleotide, siRNA, shRNA and miRNA; or, (ii) the inhibitory oligonucleotide comprises one or more modified nucleotides, wherein the one or more modified nucleotides is an LNA nucleotide; or, (iii) inhibitory oligonucleotide comprises one or more modified internucleoside linkages. 40-43. (canceled)
 44. The method of claim 26 further comprising determining that the subject has a mutant allele of the X-linked gene.
 45. The method of claim 26 further comprising determining that delivery of the XCIF inhibitor to the cell results in (i) induced expression of the X-linked gene, or a wild-type allele of the X-linked gene; or, (ii) determining that an X-chromosome is reactivated; or, (iii) determining that there is decreased expression or activity of XIST. 46-48. (canceled) 